Preparations of hydrophobic therapeutic agents, methods of manufacture and use thereof

ABSTRACT

The present invention further provides method of preparing nanocrystals of a hydrophobic therapeutic gent such as fluticasone or triamcinolone, pharmaceutical compositions (e.g., topical or intranasal compositions) thereof and methods for treating and/or preventing the signs and/or symptoms of disorders such as blepharitis, Meibomian gland dysfuntion or skin inflammation or a respiratory disease (e.g., asthma).

RELATED APPLICATIONS

This application is a Continuation of U.S. Non-Provisional applicationSer. No. 16/203,324 filed Nov. 28, 2019, which is a continuation of Ser.No. 15/660,470, filed Jul. 26, 2017, now U.S. Pat. No. 10,174,071,issued Jan. 8, 2019 which is a continuation of U.S. Non-provisionalapplication Ser. No. 14/399,780, filed Nov. 7, 2014, now U.S. Pat. No.9,822,142, issued Nov. 21, 2017, which is a national stage application,filed under 35 U.S.C. § 371, of PCT/US2013/039694, filed on May 6, 2013,which claims priority to, and the benefit of, U.S. provisionalapplication Nos. 61/644,105, filed May 8, 2012; 61/657,239, filed Jun.8, 2012; 61/692,487, filed Aug. 23, 2012, 61/763,770, filed Feb. 12,2013; and 61/788,519, filed Mar. 15, 2013, and U.S. Non-provisionalapplication Ser. No. 13/735,973, filed Jan. 7, 2013, now U.S. Pat. No.8,765,725, issued Jul. 1, 2014. The contents of each of theseapplications are hereby incorporated by reference in their entireties

FIELD OF THE INVENTION

The present invention provides a method of manufacture of sterilenanocrystals of hydrophobic therapeutic agents (such as fluticasonepropionate and triamcinolone acetonide) that are optimized to meetpharmaceutical standards of administration (e.g., topical or intranasaladministration).

BACKGROUND OF THE INVENTION

Fluticasone Propionate[(6α,11β,16α,17α)-6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-(1-oxopropoxy)androsta-1,4-diene-17-carbothioic acid, S-fluoromethyl ester], asynthetic fluorinated corticosteroid. The corticosteroids constitute aclass of primarily synthetic steroids used as anti-inflammatory andantipruritic agents. Fluticasone Propionate (FP) has been commercializedas a corticosteroid to treat inflammation associated diseases such asallergic rhinitis, asthma and atopic dermatitis. The PK/PD properties ofthis molecule have been well-established by its long standing use inhumans.

Chemically, fluticasone propionate is C₂₅H₃₁F₃O₅S. Fluticasonepropionate has a molecular weight of 500.6. It is a white to off-whitepowder and is insoluble in water. Like other topical corticosteroids,fluticasone propionate has anti-inflammatory, antipruritic andvasoconstrictive properties. The mechanism of the anti-inflammatoryactivity of the topical steroids, in general, is unclear. However,corticosteroids are thought to act by the induction of phospholipase A₂inhibitory proteins, collectively called lipocortins. It is postulatedthat these proteins control the biosynthesis of potent mediators ofinflammation such as prostaglandins and leukotrienes by inhibiting therelease of their common precursor, arachidonic acid. Arachidonic acid isreleased from membrane phospholipids by phospholipase A₂. The compoundhas potent anti-inflammatory activity and is particularly useful for thetreatment of respiratory disorders, particularly asthma. In vitro assaysusing human lung cytosol preparations have established fluticasonepropionate as a human glucocorticoid receptor agonist with an affinity18 times greater than dexamethasone, and almost twice that ofbeclomethasone-17-monopropionate (BMP), the active metabolite ofbudesonide.

Adverse reactions from the current marketed forms of fluticasonepropionate include lymphatic signs and symptoms; cardiovascularpalpitations; hypersensitivity reactions, including angioedema, skinrash, edema of the face and tongue, pruritus, urticaria, bronchospasm,wheezing, dyspnea, and anaphylaxis/anaphylactoid reactions; otitismedia; tonsillitis; rhinorrhea/postnasal drip/nasal discharge; earache;cough; laryngitis; hoarseness/dysphonia; epistaxis; tonsillitis; nasalsigns and symptoms; unspecified oropharyngeal plaques; ear, nose, andthroat polyps; sneezing; pain in nasal sinuses; rhinitis; throatconstriction; allergic ear, nose, and throat disorders; alteration orloss of sense of taste and/or smell; nasal septal perforation; blood innasal mucosa; nasal ulcer; voice changes; fluid disturbances; weightgain; goiter; disorders of uric acid metabolism; appetite disturbances;irritation of the eyes; blurred vision; glaucoma; increased intraocularpressure and cataracts; keratitis and conjunctivitis;blepharoconjunctivitis; nausea and vomiting; abdominal pain; viralgastroenteritis; gastroenteritis/colitis; gastrointestinal infections;abdominal discomfort; diarrhea; constipation; appendicitis; dyspepsiaand stomach disorder; abnormal liver function; injury; fever; toothdecay; dental problems; mouth irritation; mouth and tongue disorders;cholecystitis; lower respiratory infections; pneumonia; arthralgia andarticular rheumatism; muscle cramps and spasms; fractures; wounds andlacerations; contusions and hematomas; burns; musculoskeletalinflammation; bone and cartilage disorders; pain in joint;sprain/strain; disorder/symptoms of neck; muscular soreness/pain; achesand pains; pain in limb; dizziness/giddiness; tremors; hypnagogiceffects; compressed nerve syndromes; sleep disorders; paralysis ofcranial nerves; migraine; nervousness; bronchitis; chest congestionand/or symptoms; malaise and fatigue; pain; edema and swelling;bacterial infections; fungal infections; mobility disorders; cysts,lumps, and masses; mood disorders; acute nasopharyngitis; dyspnea;irritation due to inhalant; urticaria; rash/skin eruption; disorders ofsweat and sebum; sweating; photodermatitis; dermatitis and dermatosis;viral skin infections; eczema; fungal skin infections; pruritus; acneand folliculitis; burning; hypertrichosis; increased erythema; hives;folliculitis; hypopigmentation; perioral dermatitis; skin atrophy;striae; miliaria; pustular psoriasis; urinary infections; bacterialreproductive infections; dysmenorrhea; candidiasis of vagina; pelvicinflammatory disease; vaginitis/vulvovaginitis; and irregular menstrualcycle.

The mechanism of action of Fluticasone of all commercial andinvestigative products is identical; penetration of the plasma membraneof the cell and subsequent binding of the molecule to the cytosolicglucocorticoid receptors, represented by two separate receptors GR-α andGR-β transcribed by a single gene. Of the two receptors, GR-α isimplicated in the generation of anti-inflammatory responses. Othermechanisms of regulating inflammation are via protein—proteinsequestration via binding to other pro-inflammatory transcriptionfactors such as activator protein (AP-1), leading to the inhibition ofthe transcription of inflammatory genes. The GC-GR complex can also actindirectly via the induction of inhibitory proteins, for example IκBthat suppresses NF-κB activity. Thus, anti-inflammatory effects alsoaffect the immunological pathway, leading to immunosuppression, one ofside effects observed with the drug. Other side effects that arerelevant are ophthalmic effects such as increase of intraocular pressure(glaucoma) and the growth of cataracts. However, these side effects arecorrelated to the concentration of the drug and the route ofadministration.

A need exists for topical preparations of Fluticasone that are suitablefor ophthalmic use.

SUMMARY OF THE INVENTION

The invention is based upon the discovery of a process to preparesterile stable nanocrystals of hydrophobic drugs such as fluticasonepropionate nanocrystals or triamcinolone acetonide nanocrystals. Theprocess of the invention allows suspensions of the hydrophobic drug(e.g., fluticasone propionate and triamcinolone acetonide) nanocrystalsto be concentrated form 0.0001% to 10% while maintaining size, purity,shape (rod or plate), pH, and osmolality. This process allows theproduction of topical formulation at higher tolerable concentrationsthen has been previously achieved for the treatment of ophthalmic anddermatologic inflammatory disorders. This process also allows productionof more crystalline hydrophobic drugs and control of the sizes and sizedistributions of nanocrystals of the hydrophobic drugs. The control ofsize and size distribution may be achieved by selecting specificconditions of the process such as temperature, pH and/or viscosity ofthe component solutions for the process, type, molecular weight, and/orviscosity of the stabilizer, annealing duration, sonication outputenergy, batch size, and flow rates.

In one aspect, the invention provides a morphic form of fluticasonepropionate (Form A) characterized by an X-ray powder diffraction patternincluding peaks at about 7.8, 15.7, 20.8, 23.7, 24.5, and 32.5 degrees2θ.

The invention also provides a plurality of nanoplates of fluticasonepropionate having an average size of about 10-10000 nm, (e.g., 100-1000nm or 300-600 nm).

The invention further provides a crystalline form of purifiedfluticasone propionate, characterized by a tap density of no less than0.35 g/cm³ (e.g., no less than 0.40 g/cm³, no less than 0.45 g/cm³, noless than 0.50 g/cm³, or no less than 0.55 g/cm³).

The morphic form, crystal form, and/or nanocrystals described herein mayinclude one or more of the following features.

The morphic form is further characterized by an X-ray powder diffractionpattern further including peaks at about 9.9, 13.0, 14.6, 16.0, 16.9,18.1, and 34.3 degrees 2θ.

The morphic form is characterized by an X-ray powder diffraction patternsubstantially similar to that set forth in FIG. 31A.

The morphic form has a purity of greater than 80% by weight(e.g., >85%, >90%, >95%, >97%, >98%, or >99%).

The morphic form is further characterized by a tap density of no lessthan 0.35 g/cm³, (e.g., no less than 0.40 g/cm³, no less than 0.45g/cm³, no less than 0.50 g/cm³, or no less than 0.55 g/cm³).

The morphic form is further characterized by a melting point of 299.5°C. with a melting range of 10° C.

The morphic form is further characterized by a dissolution rate in waterof about 1 μg/g/day in water at room temperature.

The morphic form comprises fluticasone propionate nanoplates with anaverage size of about 10-10000 nm, (e.g., 100-1000 nm, 300-600 nm,400-800 nm, or 500-700 nm).

The morphic form comprises fluticasone propionate nanoplates with anarrow range of size distribution. In other words, the nanoplates aresubstantially uniform in size.

The morphic form comprises fluticasone propionate nanoplates with a sizedistribution of 50-100 nm, of 100-300 nm, of 300-600 nm, of 400-600 nm,of 400-800 nm, of 800-2000 nm, of 1000-2000 nm, of 1000-5000 nm, of2000-5000 nm, of 2000-3000 nm, of 3000-5000 nm, or of 5000-10000 nm.

The nanoplates each have a thickness between 5 nm and 500 nm (e.g.,5-400 nm, 5-200 nm, 10-150 nm or 30-100 nm).

The nanoplates have the [001] crystallographic axis substantially normalto the surfaces that define the thickness of the nanoplates.

The plurality of nanoplates is characterized by a tap density of no lessthan 0.35 g/cm³ (e.g., no less than 0.40 g/cm³, no less than 0.45 g/cm³,no less than 0.50 g/cm³, or no less than 0.55 g/cm³).

The plurality of nanoplates is characterized by a melting point of299.5° C. with a melting range of 10° C.

The plurality of nanoplates is characterized by a dissolution rate inwater of about 1 μg/g/day in water at room temperature.

The plurality of nanoplates is characterized by an X-ray powderdiffraction pattern including peaks at about 7.8, 15.7, 20.8, 23.7,24.5, and 32.5 degrees 2θ.

The plurality of nanoplates is further characterized by an X-ray powderdiffraction pattern further including peaks at about 9.9, 13.0, 14.6,16.0, 16.9, 18.1, and 34.3 degrees 2θ.

The plurality of nanoplates is characterized by an X-ray powderdiffraction pattern substantially similar to that set forth in FIG. 31A.

The plurality of nanoplates has a purity of greater than 80% by weight(e.g., >85%, >90%, >95%, >97%, >98%, or >99%).

The crystalline form is further characterized by a melting point of299.5° C. with a melting range of 10° C.

The crystalline form is further characterized by a dissolution rate inwater of about 1 μg/g/day in water at room temperature.

The crystalline form is further characterized by an X-ray powderdiffraction pattern including peaks at about 7.8, 15.7, 20.8, 23.7,24.5, and 32.5 degrees 2θ.

The crystalline form is further characterized by an X-ray powderdiffraction pattern further including peaks at about 9.9, 13.0, 14.6,16.0, 16.9, 18.1, and 34.3 degrees 2θ.

The crystalline form is characterized by an X-ray powder diffractionpattern substantially similar to that set forth in FIG. 31A.

The crystalline form has a purity of greater than 80% by weight(e.g., >85%, >90%, >95%, >97%, >98%, or >99%).

In another aspect, this invention provides a novel morphic form oftriamcinolone acetonide, i.e., Form B, which is characterized by anX-ray powder diffraction pattern including peaks at about 11.9, 13.5,14.6, 15.0, 16.0, 17.7, and 24.8 degrees 2θ.

Form B is further characterized by an X-ray powder diffraction patternincluding additional peaks at about 7.5, 12.4, 13.8, 17.2, 18.1, 19.9,27.0 and 30.3 degrees 2θ.

Form B is characterized by an X-ray powder diffraction patternsubstantially similar to the profile in red in FIG. 39 .

Form B is substantially free of impurities.

Form B has a purity of greater than 85%, greater than 90%, greater than92%, greater than 95%, greater than 96%, greater than 97%, greater than98%, or greater than 99%.

The invention also provides a method of manufacturing the plurality ofnanoplates described above. The method comprises:

providing a phase I solution (e.g., a sterile solution) comprisingfluticasone propionate and a solvent for fluticasone propionate;

providing a phase II solution (e.g., a sterile solution) comprising atleast one surface stabilizer and an antisolvent for fluticasonepropionate, wherein the at least one surface stabilizer comprises acellulosic surface stabilizer;

mixing the phase I solution and the phase II solution to obtain a phaseIII mixture, wherein sonication is applied when mixing the two solutionsand the mixing is performed at a first temperature not greater than 25°C.; and

annealing the phase III mixture at a second temperature that is greaterthan the first temperature for a period of time (T₁) such as to producea phase III suspension comprising a plurality of nanoplates offluticasone propionate.

In another aspect, the invention provides a method of manufacturingpurified, stable, sterile nanocrystals of a hydrophobic therapeuticagent. The method includes:

providing a phase I solution (e.g., a sterile solution) comprising ahydrophobic therapeutic agent and a solvent for the hydrophobictherapeutic agent;

providing a phase II solution (e.g., a sterile solution) comprising atleast one surface stabilizer and an antisolvent for the hydrophobictherapeutic agent;

mixing the phase I solution and the phase II solution to obtain a phaseIII mixture, wherein the mixing is performed at a first temperature notgreater than 25° C.; and

annealing the phase III mixture at a second temperature that is greaterthan the first temperature for a period of time (T₁) such as to producea phase III suspension comprising a plurality of nanocrystals of thehydrophobic therapeutic agent.

The methods described herein may include one or more of the followingfeatures.

The hydrophobic therapeutic agent is a steroidal drug such ascorticosteroid.

The hydrophobic therapeutic agent is fluticasone or an ester thereof ortriamcinolone acetonide.

The hydrophobic therapeutic agent is fluticasone propionate.

Sonication (e.g., with power of 10-75 W or about 50-70 W) is appliedwhen mixing the sterile phase I solution and the sterile phase IIsolution.

The first temperature is a temperature between −10° C. and 30° C.,between −10° C. and 25° C. (e.g., 22° C. or not greater than 20° C.), orbetween −5° C. and 10° C., or between 0° C. and 5° C., or between 0° C.and 2° C., or between 2° C. and 4° C., or between 2° C. and 8° C.

The second temperature is a temperature between 4° C. and 60° C., orbetween 10° C. and 40° C., or between 15° C. and 25° C.

T₁ is at least 8 hours.

At least one surface stabilizer in the phase II solution comprises acellulosic surface stabilizer.

The cellulosic surface stabilizer is methylcellulose with a molecularweight of not greater than 100 kDa.

The methyl cellulose is at a concentration of about 0.1% to 0.5% in thephase III suspension.

The cellulosic surface stabilizer used for the phase II solution is anaqueous solution.

The aqueous solution of the cellulosic surface stabilizer has aviscosity of not greater than 4000 cP (e.g., not greater than 2000 cP,not greater than 1000 cP, not greater than 500 cP, not greater than 100cP, not greater than 50 cP, not greater than 30 cP, or not greater than15 cP).

The aqueous solution of the cellulosic surface stabilizer has aviscosity of about 4 cP to 50 cP and the cellulosic surface stabilizeris methylcellulose.

The antisolvent comprises water (e.g., distilled water).

The at least one surface stabilizer in the phase II solution furthercomprises benzalkonium chloride.

The benzalkonium chloride concentration in phase II solution is about0.005% to 0.15% (e.g., about 0.01%-0.12% or 0.02%-0.08%).

The pH value of phase II solution is not greater than 6.5, or notgreater than 6.0, or not greater than 5.5.

The solvent of phase I solution comprises a polyether.

The polyether is selected from polyethylene glycol (PEG), polypropyleneglycol (PPG), and a mixture thereof.

The polyether is selected from PEG400, PPG400, and a mixture thereof.

The PEG 400 is at a concentration of about 20 to 35% in the phase Isolution.

The PPG 400 is at a concentration of about 65% to 75% in the phase Isolution.

The solvent of phase I solution comprises one or more polyols such asmonomeric polyols (e.g., glycerol, propylene glycol, and ethyleneglycol) and polymeric polyols (e.g., polyethylene glycol).

The phase I solution further comprises a surface stabilizer.

The surface stabilizer in the phase I solution is TWEEN 80® (polysorbate80), e.g., at a concentration of about 7.0% to 15% in the phase Isolution.

The volume ratio of the phase I solution to phase II solution rangesfrom 1:10 to 10:1 (e.g., 1:3 to 3:1, or 1:2 to 2:1, or about 1:1).

The cellulosic surface stabilizer is methylcellulose with a molecularweight of not greater than 100 kDa, the first temperature is atemperature between 0° C. and 5° C., the second temperature is atemperature between 10° C. and 40° C., and T₁ is at least 8 hours.

The method further comprises purification of the plurality ofnanocrystals of the hydrophobic therapeutic agent by tangential flowfiltration or by continuous flow centrifugation. The method may furthercomprise drying the plurality of nanocrystals of the hydrophobictherapeutic agent by, e.g., filtration, vacuum drying, orcentrifugation. The method may further comparing, after purifying thenanocrystals by, e.g., centrifugation, mixing the purified nanocrystalswith a suitable aqueous solution to which additional excipients can beadded to form a final formulation that meets FDA criteria for ophthalmicor dermatologic administration. For example, the mixing is performed ina mixer (e.g., a SILVERSON® Lab Mixer) at room temperature at 6000 RPMfor about 60 mins or longer.

Purified, stable, sterile nanocrystals of fluticasone by mixing asterile phase I solution of fluticasone with a sterile phase II solutioncomprising benzalkonium chloride, methyl cellulose, and distilled watersuch as to produce a phase III suspension containing a suspension offluticasone nanocrystals. The nanocrystals are between 400-800 nm. Topurify, i.e., to remove and or reduce the concentration ofcrystallization solvents of the phase I and phase II solution, thefluticasone nanocrystals are washed and exchanged into a suitableaqueous solution. The exchanging is performed for example by usingtangential flow filtration (TFF) or hollow fiber filter cartridge. Insome aspects the nanocrystals are exchange into a formulation that meetsFDA criteria for ophthalmic or dermatologic administration.Alternatively the nanocrystals are exchanged into a sterile aqueoussolution to which additional excipients are added to form a finalformulation that meets FDA criteria for ophthalmic or dermatologicadministration. The concentration of fluticasone in the final aqueousbuffer solution is about between 0.0001% to 10% (w/v). In some aspectsan annealing step is performed before the buffer exchanging step. Theannealing step is performed at about 25-40° C. and is for a duration ofbetween about 30 minutes to 24 hours.

Preferably, the fluticasone of the phase I solution is at aconcentration of about 0.4% to 1.0% w/v. More preferably, thefluticasone of the phase I solution is at a concentration of about 0.45%w/v.

In some aspects the phase I solution further contains TWEEN 80®(polysorbate 80), polyethylene glycol (PEG) 400 and polypropylene glycol(PPG) 400. The TWEEN 80® (polysorbate 80) is at a concentration of about7.0% to 15% w/v. The PEG 400 is at a concentration of about 20 to 35%(w/v). The PPG 400 is at a concentration of about 65% to 75% (w/v). In apreferred embodiment, the phase I solution contains fluticasone at aconcentration of about 0.45% w/v, TWEEN 80® (polysorbate 80) at aconcentration of about 7.44%, PEG 400 at a concentration of about 23%(w/v) and PPG 400 at a concentration of about 69.11% (w/v).

The mixing of phase I and phase II is performed at a temperature notgreater than 8° C. (e.g., 0-2° C., 2-4° C., or 2-8° C.). The volumeratio of phase I to phase II is 0.15 to 0.3 or 1:1 to 1:3. The phase Isolution is mixed with the phase II solution at a flow rate of 0.5 to1.4 ml/min, wherein the phase II solution is stationary. See, e.g., FIG.3 . In other embodiments the phase III is formed in a flow reactor bycombining the phase I solution at a flow rate of 0.5-900 ml/min (e.g.,0.5-2.0 ml/min, 10-900 ml/min, 12-700 ml/min, 50-400 ml/min, 100-250ml/min, or 110-130 ml/min) and the phase II solution at a flow rate of2.5-2100 ml/min (e.g., 2.5-10 ml/min, 10-900 ml/min, 12-700 ml/min,50-400 ml/min, 100-250 ml/min, or 110-130 ml/min). See, e.g., FIG. 4 .In some embodiments, the flow rate of phase I and that of phase IIsolutions are substantially the same. In other embodiments, the flowrate of phase I is less than that of phase II, e.g., volume ratio of thephase I solution to phase II solution is about 1:2 or 1:3. In someembodiments, the flow rate of the phase III suspension coming out of aflow reactor is at about 20-2800 ml/min (e.g., about 100-800 ml/min or200-400 ml/min). Optionally, the phase III mixture is sonicated.

In some embodiments the final aqueous buffer comprising methylcellulose, a permeation enhancer and a wetting agent. The methylcellulose is for example at a concentration of about 0.5% (w/v).

Also included in the invention is a plurality of the nanocrystalsproduced by the methods of the invention and compositions (e.g., apharmaceutical composition) containing the nanocrystals. The compositionis substantially free of organic solvents. The nanocrystals have anaverage size ranging between 400-800 nm (e.g., 300-600 nm, 400-600 nm,or 500-700 nm). The nanocrystals do not agglomerate and do not increasein size over a period of 24 hours. The nanocrystals are nanoplates,e.g., fluticasone propionate nanoplates having the [001]crystallographic axis substantially normal to the surfaces that definethe thickness of the nanoplates. The nanoplates can have a thicknessranging from about 5 nm to 100 nm. Optionally, the nanocrystals arecoated with methyl cellulose.

Further provided by the invention is a sterile topical nanocrystalfluticasone formulation containing a suspension of between 0.0001%-10%w/v fluticasone nanocrystals of the invention and a pharmaceuticallyacceptable aqueous excipient. In some aspects the formulation has aviscosity between 10-20 cP at 20° C. The osmolality of the formulationis about 280-350 mOsm/kg. The pH of the formulation is about 6-7.5.

In another aspect the invention provides a method of treating oralleviating a symptom of an ocular disorder (e.g., blepharitis,meibomian gland dysfunction, post operative pain or post-operativeocular inflammation, dry eye, eye allergy, or uveitis) by administering,e.g., topically to the lid margin, skin, or ocular surface of, a subjectin need thereof an effective amount of the formulations (e.g., topicalformulations) of the invention. The formulation is administered forexample by using an applicator (e.g., a brush or swab). In oneembodiment, a therapeutically effective amount of the formulation isadministered to a subject in need thereof for treating blepharitis, viae.g., an applicator (e.g., a brush such as LATISSE® (bimatoprostophthalmic solution) brush or a swab such as 25-3317-U swab). In someembodiments, the formulation is a sterile topical nanocrystalfluticasone propionate formulation containing a suspension of between0.001%-5% FP nanocrystals of the invention (e.g., 0.01-1%, or about0.25%, 0.1%, or 0.05%), and a pharmaceutically acceptable aqueousexcipient. In some embodiments, the formulation further contains about0.002-0.01% (e.g. 50 ppm±15%) benzalkonium chloride (BKC). In someembodiments, the formulation further contains one or more coatingdispersants (e.g., TYLOXAPOL*(formaldehyde, polymer with oxirane and4-(1,1,3,3-tetrametylbutyl)phenorl), polysorbate 80, and PEG stearatesuch as PEG40 stearate), one or more tissue wetting agents (e.g.,glycerin), one or more polymeric stabilizers (e.g., methyl cellulose4000 cP), one or more buffering agents (e.g., dibasic sodium phosphateNa₂HPO₄ and monobasic sodium phosphate NaH₂PO₄, and/or one or moretonicity adjusting agents (e.g., sodium chloride). In some embodiments,the formulation has a viscosity between 40-50 cP at 20° C. In someembodiments, the osmolality of the formulation is about 280-350 (e.g.,about 285-305) mOsm/kg. In some embodiments, the pH of the formulationis about 6.8-7.2. In some embodiments, the formulation has a viscositybetween 40-50 cP at 20° C. In some embodiments, the FP nanocrystals inthe formulation have a median size of 300-600 nm, a mean size of 500-700nm, a D50 value of 300-600 nm, and/or a D90 value of less than 2 μm(e.g., less than 1.5 μm).

In yet another aspect the invention provides a method of treating oralleviating a respiratory disease (e.g., asthma or chronic obstructivepulmonary disease (COPD)), rhinitis, dermatitis, or esophagitis byadministering to a subject in need thereof an effective amount of thepharmaceutical composition of the invention.

Also provided is a pharmaceutical composition comprising one or morepharmaceutically acceptable carriers or excipients and the nanocrystalsof hydrophobic drugs (e.g., fluticasone propionate) produced by themethods of the invention. The composition can be in the form of drypowder/inhalers, ophthalmic preparations, sprays, ointments, creams,pills, etc.

In a further aspect the invention provides a semi-flexible polyurethaneapplicator comprising fluticasone nanocrystals of the invention and apharmaceutically acceptable aqueous excipient.

In yet another aspect, the invention provides a surgical or implantabledevice (e.g., a stent, angioplasty balloon, catheter, shunt, accessinstrument, guide wire, graft system, intravascular imaging device,vascular closure device, endoscopy accessory, or other device disclosedherein) coated or impregnated with the fluticasone propionate crystalsof the invention. In some embodiments, coating or embedding fluticasonepropionate crystals into a surgical or implantable device modifies therelease time of the drug. For example, coating or embedding fluticasonepropionate crystals into a surgical or implantable device extends therelease time of the drug.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Advantages of the methods of the invention include that the product(e.g., nanocrystals of the hydrophobic drug) is purer (or at least notless pure), is more crystalline, and/or is more stable than stockmaterial of the drug. The advantages also include that the size and sizedistribution of the product are controllable and the product's size canbe substantially uniform (which may lead to better control of drugrelease in vivo), and that the methods of the invention cause little orno degradation to the drug. Other features and advantages of theinvention will be apparent from and encompassed by the followingdetailed description and claims.

BRIEF DESCRIPTIONS OF FIGURES

FIG. 1 is a summary of physical and chemical characteristics offluticasone propionate.

FIG. 2 is a HPLC chromatogram of fluticasone propionate and its commonimpurities.

FIG. 3 is a scheme of an embodiment of the process of the invention(denoted as “batch process”).

FIG. 4 is a scheme of another embodiment of the process of the invention(denoted as “flow process”).

FIG. 5 is a plot showing that average sizes of fluticasone propionatenanocrystals are controllable by changing specific compositions of phaseII solution.

FIG. 6 is a plot showing particle sizes of fluticasone propionateproduced by top-down techniques such as microfluidization, jet-milling,ultrasound sonication (wet milling) and homogenization.

FIG. 7 is a plot showing the effect of pH of phase II solution onparticle size of fluticasone propionate.

FIG. 8 is a plot showing the effect of different stabilizers in phase IIsolution on particle size of fluticasone propionate.

FIG. 9 is a plot showing the effect of pH of phase III mixture onparticle size of fluticasone propionate.

FIG. 10 is a plot showing that purified fluticasone propionatenanocrystals do not aggregate over time.

FIG. 11 is a plot showing the effect of temperature when mixing thephase I and phase II solutions on particle size of fluticasonepropionate.

FIG. 12 is a plot showing the effect of annealing temperature and onparticle size of fluticasone propionate with concentration of 0.1% inthe phase III suspension.

FIG. 13 is a plot showing the effect of annealing temperature and onparticle size of fluticasone propionate with concentration of 10% in thephase III suspension.

FIG. 14 is a plot showing the effect of filter type on loss of drugcrystals.

FIG. 15 is a plot showing the effect of filter pore size on loss of drugcrystals.

FIG. 16 is a plot showing the dispersibility of formulations as afunction of batch scale (from left to right: 20 g, 100 g, 250 g, 1000 g,and 2000 g).

FIG. 17 is a plot showing the dispersibility of formulations as afunction of FP concentration (from left to right: 10%, 5%, 1%, 0.1%,0.05%, 0.01%, and 0.005%).

FIG. 18 is a plot showing the uniformity of formulation as a function oftime.

FIG. 19 is a scheme of a flow reactor.

FIG. 20 is a plot showing the effect of flow rates on particle size offluticasone propionate in the flow process.

FIGS. 21A-C are plots showing particle size distributions of FPnanocrystals made by the batch process, FP particles made byhomogenization, and FP stock received from manufacturer.

FIGS. 22A-D is a group of plots showing stability of particle size ofthe fluticasone propionate nanosuspension, at 25° C. and 40° C. for upto 75 days.

FIG. 23 is a plot showing dissolution rates of fluticasone propionatehomogenized (1-5 microns, represented by grey square dots) andfluticasone propionate crystals produced by the batch process (400-600nm, represented by black diamond dots).

FIGS. 24A and 24B are chromatograms of fluticasone propionate stockmaterial and nanocrystals produced by the batch process respectively.

FIGS. 25A and 25B are optical micrographs (Model: OMAX, 1600×) of driedfluticasone propionate crystals prepared by the batch process and FPstock material, respectively.

FIGS. 26A and 26B are Scanning Electron Micrographs of dried fluticasonepropionate crystals prepared by the batch process.

FIGS. 27A and 27B are Scanning Electron Micrographs of dried fluticasonepropionate stock material and FP crystals prepared by homogenizationrespectively.

FIGS. 28A and 28B are combined DSC/TGA of fluticasone propionatenanocrystals produced by the batch process and FP stock material,respectively.

FIGS. 29A and 29B are Fourier Transform Infrared Spectroscopic Scan ofFP nanocrystals produced by the batch process of the invention.

FIGS. 30A and 30B are Fourier Transform Infrared Spectroscopic Scan ofFP stock material.

FIG. 31A is XRPD pattern of fluticasone propionate nanocrystals producedby the batch process (black).

FIG. 31B is XRPD pattern of fluticasone propionate nanocrystals producedby the batch process (black) overlaid with the calculated XRPD patternof polymorph 1 (red) and polymorph 2 (blue) overlaid. The blue arrowsshow some of the differences in the XRPD patterns.

FIG. 32 is a plot showing size distribution of triamcinolone acetonidecrystals produced by the methods of the invention.

FIG. 33 is DSC scan of triamcinolone acetonide stock material.

FIG. 34 is DSC scan of triamcinolone acetonide crystals produced by themethods of the invention.

FIG. 35 is thermogravimetric analysis of triamcinolone acetonide stockmaterial.

FIG. 36 is thermogravimetric analysis of triamcinolone acetonidecrystals produced by the methods of the invention.

FIGS. 37A-E are Scanning Electron Micrographs of triamcinolone acetonidestock material and triamcinolone acetonide crystals prepared by themethods of the invention at different magnifications: A andB-triamcinolone acetonide stock material at 100× and 5000×magnifications respectively; C, D, and E-triamcinolone acetonidecrystals produced by the methods of the invention at 100×, 5000× and10,000× magnifications respectively.

FIG. 38 is a schematic showing an embodiment of the process of theinvention for production and purification process for fluticasonepropionate nanocrystals.

FIG. 39 is XRPD pattern of triamcinolone acetonide nanocrystals preparedby the methods of the invention (red) overlaid with the XRPD pattern oftriamcinolone acetonide stock material (blue). The arrows show some ofthe differences in the XRPD patterns.

FIG. 40 is a plot showing the linearity of fluticasone propionate informulation vehicle as set forth in Table 5.

FIG. 41 is a plot showing the linearity of fluticasone propionate usingmobile phase as a diluent as set forth in Table 6.

DETAILED DESCRIPTION OF THE INVENTION

The invention describes methods and compositions to produce sterilenanocrystals (optionally nanosuspensions) of hydrophobic therapeuticagents (such as fluticasone propionate) that are optimized to meetpharmaceutical standards of administration (e.g., topical or intranasaladministration). The compositions produced by the methods are ideallysuited for the topical treatment of inflammatory disorders such asophthalmic disorders and dermatologic disorders. The compositionsproduced by the methods are also ideally suited for systemic ornon-systemic treatment of disorders that the hydrophobic drugs in thecompositions are used for, such as inflammatory disorders, respiratorydisorders, autoimmune diseases, and cancer.

The drug nanocrystals made by the methods of the invention, whenadministered to a subject in need thereof, can be in various forms thatare suitable for the specific route of administration, e.g. the form ofeye drops, gels, ointments, dry powers, gels, aerosols, or a colloidalsuspension (e.g., a liquid suspension). For example, the drugnanocrystals are the “dispersed” phase, suspended in another phase whichis the “continuous” phase. A nanosuspension can be defined as colloidaldispersions of nano-sized drug particles that are produced by a suitablemethod and stabilized by a suitable stabilizer or surface stabilizer.Unless otherwise specified, the terms “stabilizer,” “surfacestabilizer,” and “steric stabilizer” are used interchangeably herein. Inone embodiment, the drug is delivered or formulated for delivery via asystemic or local route. For example, the drug is delivered orformulated for delivery directly or via an applicator (e.g., a brush orswab). For example, the drug is delivered or formulated for delivery viaa local route to a tissue, such as an ocular tissue and/or adnexa. Thedrug can be delivered or formulated for delivery via intraocular,intravitreal, subretinal, intracapsular, suprachoroidal, subtenon,subconjunctival, intracameral, intrapalpebral, cul-d-sac retrobulbar, orperibulbar injections. The drug can also be delivered or formulated fordelivery via topical application to a tissue, such as an ocular tissueand/or adnexa. The drug can also be delivered or formulated for deliveryvia an implantable or surgical (e.g., drug delivery) device.

Nanosuspensions, such as nanocrystal suspensions, of insoluble drugs candramatically lower its effective concentration by enhancingbioavailability. By “bioavailable” is meant dissolved drug that ismolecularly available for absorption by cells.

Fluticasone propionate is almost insoluble in water with a solubility of0.14 micrograms/ml. Since most ophthalmic suspensions are aqueous, theparticle size of an insoluble drug determines its rate of dissolutioninto dissolved drug (or, bioavailable drug) at any given time. One wayto enhance bioavailability is to ensure a completely dissolved drugsolution. For insoluble drugs, the way to enhance the bioavailability ofa water-insoluble drug is by utilization of micronized or nanosizeddosage forms. In the case of fluticasone propionate, the rate ofdissolution is dramatically enhanced by lowering the particle size. Therelease rate of fluticasone propionate particles of size 800-900 nm ismany-fold that of that of particles >10 microns. Thus, nanosuspensionsof fluticasone propionate have the potential to yield potent medicationsthat are effective at concentrations that do not cause adverse sideeffects. At higher concentrations, fluticasone propionate can causeelevation of intraocular pressure leading to glaucoma and cataracts. Aneffective formulation of fluticasone propionate can be envisioned atlower concentrations, if the drug is nanoparticulate, or or in a morphicform that is more water-soluble. For fluticasone propionate, theeffective concentration in commercialized drug products range from0.005% (e.g., CUTIVATE®) and 0.5% (e.g., FLONASE®). Thus, rendering adrug “effective” at concentrations not previously contemplated for thatindication would be a surprising and unexpected result. Similarly, fortriamcinolone acetonide, another hydrophobic drug (with a watersolubility of 17.5 μg/mL at 28° C.), when the drug is nanoparticulateform generated e.g., via the methods of the invention, an effectiveformulation of TA can be obtained at unexpectedly lower concentrationsof TA not previously contemplated for a particular indication.

Thus, in the design of topical medications that require immediaterelief, then sustained relief, it is surmised that a nanocrystalinesuspension that is also bioadhesive, will assist in enhancing theresidence time of the drug, while increasing the bioavailability at thesame time. In the examples described in this invention, fluticasonepropionate suspensions were developed for the treatment of blepharitis,which is characterized by inflammation and infection of the eyelid.However, the fluticasone propionate compositions described herein canalso be utilized for the prevention or treatment of other ophthalmicinflammatory conditions. For example, the compositions described in theinvention can be used for post-operative care after surgery. Forexample, the composition of the invention can be used to control of painafter surgery, control of inflammation after surgery, argon lasertrabceuloplasty and photorefractive procedures. Furthermore, thefluticasone propionate compositions can be used to treat otherophthalmic disorders such as ophthalmic allergies, allergicconjunctivitis, cystoid macular edema uveitis, or meibomian glanddysfunction. Additionally, the fluticasone propionate compositions canbe used to treat dermatologic disorders such as atopic dermatitis,dermatologic lesion, eczema, psoriasis, or rash.

Challenges of Stable Nanocrystal Fabrication of Hydrophobic Drugs

The successful fabrication of nanosuspensions has two major challenges.The first challenge is the generation of particles that are of thedesired size. For most drugs that are insoluble in water, the desiredparticle size is submicron, ranging from the low nm to the high (10-990nm). The second step is maintaining particle size long-term. Both stepsare challenging.

Drug suspensions are normally prepared by “top-down” techniques, bywhich the dispersion is mechanically broken into smaller particles.Techniques such as wet milling, sonication, microfluidization and highpressure homogenization are examples of this technique to createmicronized and nanosized particles. In high pressure homogenization, thenanocrystal size resulting from the process depends not only on thehardness of the drug material but also on the homogenization pressureand cycle number. It does not, however, depend on the type ofstabilizer. Thus, the efficiency of the stabilizer—whether or not it isable to prevent aggregation of the particles—is shown after processingand during storage. Accordingly, it is extremely important to understandthe phenomena involved in particle formation in the particular processused.

During milling or mechanical particle size reduction methods, twoopposite processes are interacting in the milling vessel: fragmentationof material into smaller particles and particle growth throughinter-particle collisions. The occurrence of these two oppositephenomena is dependent on the process parameters. Often after a certaintime-point, the particle size has achieved a constant level andcontinuing the milling does not further decrease the particle size. Insome cases an increase in grinding time may even lead to a gradualincrease of particle size and heterogeneity of the material, whiledecreased particle sizes are achieved with decreased milling speeds.Changes in the physical form or amorphization are also possible duringthe milling. Mechanical pressure above certain critical pressure valuesincreases lattice vibrations, which destabilize the crystal lattice. Thenumber of defects increases and transformation into an amorphous stateoccurs above a critical defection concentration. The high stresses onthe drug crystals during particle reduction techniques result indestabilization of the crystal structure, loss in crystallinity andsometimes, shift to less stable polymorphic forms. Creation of amorphousregions in the crystalline structures leads to gradual increase inparticle size as the suspension shifts back into a stable, crystallinemorphology.

Another challenge for nanocrystal fabrication is gradual growth in thesize of the particles, also called “Ostwald Ripening”. Crystal growth incolloidal suspensions is generally known as Ostwald ripening and isresponsible for changes in particle size and size distribution. Ostwaldripening is originated from particles solubility dependence on theirsize. Small crystals have higher saturation solubility than larger onesaccording to Ostwald-Freundlich equation, creating a drug concentrationgradient between the small and large crystals. As a consequence,molecules diffuse from the higher concentration surrounding smallcrystals to areas around larger crystals with lower drug concentration.This generates a supersaturated solution state around the largecrystals, leading to drug crystallization onto the large crystals. Thisdiffusion process leaves an unsaturated solution surrounding the smallcrystals, causing dissolution of the drug molecules from the smallcrystals into the bulk medium. This diffusion process continues untilall the small crystals are dissolved. The Ostwald ripening isessentially a process where the large particles crystals at the expenseof smaller crystals. This subsequently leads to a shift in the crystalssize and size distribution of the colloidal suspension to a higherrange. Dispersions with dissolved drug in the continuous phase alsoinvariably lead to instability in particle size.

Another challenge with nanocrystals is agglomeration, or clumping ofparticles. The stabilizer plays a critical role in stabilizing thedispersion. The stabilizer needs to adsorb on the particle surfaces inorder for proper stabilization to be achieved. Furthermore, theadsorption should be strong enough to last for a long time. Adsorptionof the stabilizer may occur by ionic interaction, hydrogen bonding, vander Waals or ion-dipole interaction or by hydrophobic effect.

Possible interactions between the functional groups of a stabilizer anddrug materials always need to be considered before selecting thedrug-stabilizer pair. Many drugs have structures containingfunctionalities like phenols, amines, hydroxyl groups, ethers orcarboxylic acid groups, which are capable of interactions. Strong ionicinteractions, hydrogen bonding, dipole-induced forces, and weak van derWaals or London interactions may enhance or disturb particle formation.The concentration level of the stabilizer is also important. Theadsorption/area is a surface property that does not usually depend onparticle size. As the adsorbed amount correlates to the surface area,this means that the total amount of stabilizer is directly related tothe crystals size. Adsorption of polymer molecules onto the crystalssurfaces takes place when the free energy reduction due to theadsorption compensates the accompanying entropy loss. Because stericstabilization is based on adsorption/desorption processes, processvariables such as the concentration of the stabilizer, particle size,solvent, etc. are important factors for the effectiveness of thestabilizer.

Another way to stabilize the crystals size has been in the spray-dryingof the particulate suspension in the presence of specific stabilizers, atechnique that has been used to generate aerosolized microparticles offluticasone propionate. Combinations of top-down methods are also usedto generate particles of the desired size. Yet another method tostabilize the particle size has been to lyophilize the particulatesuspension.

The other method commonly used to create nanosuspensions is theantisolvent precipitation method, whereupon a drug solution isprecipitated as nanocrystals in an antisolvent. This approach is calledthe “bottom-up” crystallization approach, whereupon the nanocrystals areproduced in-situ. The precipitation of the drug as nanocrystals isusually accompanied by homogenization or sonication. If the drug isdissolved in an organic solvent such as acetone prior to precipitation,the organic solvent has to be removed after formation of the particles.This is usually performed by evaporation of the solvent. Thisevaporative step poses challenges to this method of particle formation,since the process of evaporation can alter the dynamics of particlestabilization, often seen as rapid increases in particle size.Furthermore, residual levels of organic solvents often remain bound toexcipients used in the formulation. Thus, this method, though explored,has its challenges and is generally not preferred.

The nanocrystals of a hydrophobic drug produced by the process definedin this invention do not use toxic organic solvents that need removaland do not display particle instability defined in the sections above.

Core Features the Invention

This invention provides a sonocrystallization/purification process thatcan produce nanocrystals of a drug (e.g., a hydrophobic drug) orsuspensions containing the nanocrystals. The process: (a) incorporatessterile filtration of all components prior to production of thenanocrystals, (b) produces the crystals at the desired size, (c)stabilizes the nanocrystals by the use of specific steric stabilizingcompositions, in combination with annealing at specific temperatures,(d) provides the formulator the flexibility to purify the particles byreplacing the original continuous phase with another continuous phaseand (d) provides the flexibility to achieve a final desiredconcentration of drug in the final formulation vehicle. In step (d), thesignificance of the purification step may be a key and critical aspectof the invention, since the composition that produces and stabilizes theparticles at a desired size is nuanced and dependent upon parameters ofionic strength, polymer molecular weight and structure and pH. Thecomposition used to create the particles is usually not the compositionthe formulator envisions as the final formulation, or the final drugconcentration. This is addressed by spray-drying, or lyophilization. Thenanocrystals produced by this process are of the size range 100 nm-500nm, 500-900 nm, 400-800 nm and 900 nm to 10000 nm. Preferably thenanocrystals are of the size range of 400-800 nm (e.g., 400-600 nm). Thesize and size distribution of nanocrystals of the invention can bedetermined by conventional methods such as dynamic light scattering(DLS), scanning electron microscopy (SEM), transmission electronmicroscopy (TEM), and X-ray Power Diffraction (XRPD). In this invention,the nanocrystals are purified by exchange with the final biocompatible,tissue-compatible buffer.

Two-Part Process: The process is characterized by a two-part process toprepare nanocrystals, defined as Step 1 and Step 2. Optionally, theprocess is a single step, whereupon the final formulation is prepared ina single step (only, Step 1). For the two-step process (Step 1, followedby Step 2), the first part of the process is nanocrystal production atthe desired size (Step 1). The second part of the process is nanocrystalpurification to yield highly pure nanocrystals suspended at the desireddrug concentration and optimized excipient composition for the finalformulation (Step 2).

Drug Concentrations: In a preferred embodiment, the initial nanocrystalconcentration (after Step 1) is at 0.1% drug (e.g., a corticosteroidsuch as FP), but the final formulation may be as high as 10% (after Step2). The initial concentration of the suspension may be less than 0.1%(in Step 1) and concentrated to 10% during the purification process(Step 2) with the same vehicle composition, or a different vehiclecomposition. The initial concentration of the suspension may be 0.1% orless than 0.1%, preferably 0.06%. The initial suspension may be purifiedto a lower concentration (in Step 2) with the same vehicle composition,or a different vehicle composition. In a preferred composition, theinitial suspension may be formed at 0.06% (in Step 1) and purified to0.06% or lower (in Step 2) with the same initial vehicle composition, ora different vehicle composition. The initial concentration of thenanosuspension may be 1%, 1%-0.5%, 0.5%-0.1%, 0.1%-0.05%, 0.05%-0.01%,0.01%-0.005%, 0.005%-0.001%, 0.001%-0.0005%, 0.0005%-0.0001%,0.0001%-0.00001%.

Step 1 comprises dissolution of the drug in FDA-approved excipients tocreate Phase I. The solution (Phase I) is then sterile filtered througha 0.22 micron PVDF (polyvinylidene fluoride) filter. A solutioncontaining a specific composition of a steric stabilizer at certainviscosity, pH and ionic strength is prepared. This is Phase II. In oneembodiment, the drug is a steroidal drug. In a preferred embodiment, thedrug is fluticasone propionate. In another preferred embodiment, thedrug is fluticasone furoate. In another embodiment, the drug is any saltform of fluticasone propionate.

In one embodiment, Step 1 includes:

providing a phase I solution (e.g., a sterile solution) comprising ahydrophobic therapeutic agent and a solvent for the hydrophobictherapeutic agent;

providing a phase II solution (e.g., a sterile solution) comprising atleast one surface stabilizer and an antisolvent for the hydrophobictherapeutic agent;

mixing the phase I solution and the phase II solution to obtain a phaseIII mixture, wherein the mixing is performed at a first temperature notgreater than 25° C.;

annealing the phase III mixture at a second temperature that is greaterthan the first temperature for a period of time (T₁) such as to producea phase III suspension comprising a plurality of nanocrystals of thehydrophobic therapeutic agent, and

optionally purifying the nanocrystals by, e.g., tangential flowfiltration, hollow fiber cartridge filtration, or centrifugation (e.g.,continuous flow centrifugation).

Optionally, centrifugation is performed at about 1.6 L/min at about39,000 xg.

Optionally, Step 1 includes a dilution step with a solution followingthe annealing step and prior to the purification step. For example, thedilution step includes re-dispersing the nanocrystals in a solution. Thesolution used for dilution can include about 0.002-0.01% (e.g. 50ppm±15%) benzalkonium chloride, 0.01-1% polysorbate 80 (e.g., about0.2%), 0.01-1% PEG40 stearate (e.g., about 0.2%), buffering agent (e.g.,citrate buffer, pH 6.25), and water. A pellet formed during purification(e.g., during centrifugation) is re-dispersed into a final formulation(see, e.g., FIG. 38 ). The pellet can be added into a suitable aqueoussolution to redisperse the nanocrystals contained in a mixer (e.g., aSILVERSON® Lab Mixer). The redisperion can be performed at roomtemperature at 6000 RPM for about 45 mins or longer (e.g., about 60 minsor longer) to obtain a final formulation that meets FDA criteria forophthalmic or dermatologic administration. The formulation may containone or more pharmaceutically acceptable excipients.

For example, the hydrophobic therapeutic agent is a steroid.

For example, the hydrophobic therapeutic agent is fluticasone propionateor triamcinolone acetonide.

For example, the at least one surface stabilizer comprises a cellulosicsurface stabilizer such as methyl cellulose.

For example, the methyl cellulose has a molecular weight of not greaterthan 100 kDa.

For example, the cellulosic stabilizer (e.g., methyl cellulose) used forthe phase II solution has a viscosity between 4 cP and 50 cP, e.g.,15-45 cP.

For example, the first temperature is, e.g., not greater than 20° C.,not greater than 8° C., e.g., <4° C., or <2° C. or 0-4° C.

For example, the second temperature, i.e., the annealing temperature, isbetween 20° C. and 60° C.

For example, the annealing step is necessary for decreasing the particlesize of the nanocrystals and/or for hardening the nanocrystals (e.g., toincrease to hardness of the nanocrystals).

For example, continuous flow centrifugation is performed at about 1.6L/min at about 39,000×g.

For example, the nanocrystals produced by the methods described hereinhave an average size between 10 nm and 10000 nm (e.g., 50-5000 nm,80-3000 nm, 100-5000 nm, 100-2000 nm, 100-1000 nm, or 100-800 nm).

For example, the nanocrystals produced by the methods described hereinhave a particle size suitable for delivery by micro needles (i.e., 27-41gauge). For example, when injected in the suprachoroidal space of theeye, the nanocrystals can be efficiently delivered to the back of theeye or will dissolve more slowly so that the drug treats target tissueswithout leeching into front-of-eye tissues, such as lens, ciliary body,vitreous, etc., thereby minimizing ocular side effects, such as highintraocular pressure (IOP) or cataract formation.

For example, the nanocrystals produced by the methods described hereinhave a narrow range of size distribution. In other words, thenanocrystals are substantially uniform in size.

For example, the ratio of the nanocrystals' D90 and D10 values is lowerthan 10, e.g., lower than 5, lower than 4, lower than 3, lower than 2,or lower than 1.5. For example, the nanocrystals have a sizedistribution of 50-100 nm, of 100-300 nm, of 300-600 nm, of 400-600 nm,of 400-800 nm, of 800-2000 nm, of 1000-2000 nm, of 1000-5000 nm, of2000-5000 nm, of 2000-3000 nm, of 3000-5000 nm, or of 5000-10000 nm.

For example, the nanocrystals produced by the methods described hereinhave D90 value of not greater than 5000 nm (e.g., not greater than 4000nm, not greater than 3000 nm, not greater than 2000 nm, not greater than1000 nm, not greater than 900 nm, not greater than 800 nm, not greaterthan 700 nm, not greater than 700 nm, not greater than 600 nm, notgreater than 500 nm, not greater than 400 nm, not greater than 300 nm,not greater than 200 nm, not greater than 100 nm, or not greater than 80nm).

For example, the nanocrystals produced by the methods described hereinare coated with methyl cellulose.

For example, the methyl cellulose-coated nanocrystals produced by themethods described herein are stable, e.g., they do not aggregate.

For example, the nanocrystals produced by the methods described hereinare fluticasone propionate nanocrystals having a size distribution of400-600 nm.

For example, the nanocrystals produced by the methods described hereinare triamcinolone acetonide nanocrystals having a size distribution of300-400 nm.

For example, the nanocrystals produced by the methods described hereinare either in the form of a liquid suspension or dry powder.

For example, the nanocrystals produced by the methods described hereinhave a concentration of from 0.0001% to 10%, to 20%, to 30%, to 40%, to50%, to 60%, to 70%, to 80%, to 90%, to 99%, or to 99.99%.

For example, sonication is applied when mixing the phase I and IIsolutions.

For example, the methyl cellulose is at a concentration ranges from 0.1%to 0.5% (e.g., 0.2-0.4%) in the phase II solution.

For example, the phase II solution further includes a second stabilizer,e.g., benzalkonium chloride at a concentration ranges from 0.005% to0.1% (e.g., 0.01-0.02%).

For example, the phase II solution has pH of 5.5 when the hydrophobicdrug is fluticasone propionate.

For example, the phase II solution has pH of about 4 when thehydrophobic drug is triamcinolone acetonide.

For example, the solvent of phase I solution comprises a polyether.

For example, the polyether is selected from polyethylene glycol (PEG),polypropylene glycol (PPG), and a mixture thereof.

For example, the polyether is selected from PEG400, PPG400,PEG40-stearate, and a mixture thereof.

For example, the PEG 400 is at a concentration of about 20 to 35% in thephase I solution.

For example, the PPG 400 is at a concentration of about 65% to 75% inthe phase I solution.

For example, the solvent of phase I solution comprises one or morepolyols such as monomeric polyols (e.g., glycerol, propylene glycol, andethylene glycol) and polymeric polyols (e.g., polyethylene glycol).

For example, the solvent of phase I solution comprises one or moremonomeric polyols.

For example, the phase I solution further comprises a surfacestabilizer.

For example, the surface stabilizer in the phase I solution is TWEEN 80®(polysorbate 80), e.g., at a concentration of about 7.0% to 15% in thephase I solution.

For example, the concentration of hydrophobic drug in the phase Isolution is about 0.1-10%, e.g., 0.1 to 5.0%, 0.2-2.5%, or 0.4 to 10%.

For example, when the hydrophobic drug is FP, the concentration of FP inthe phase I solution is about 0.1-10%, e.g., 0.4 to 1.0%.

For example, the volume ratio of the phase I solution to phase IIsolution ranges from 1:10 to 10:1 (e.g., 1:3 to 3:1, or 1:2 to 2:1, orabout 1:1).

For example, the cellulosic surface stabilizer is methylcellulose with amolecular weight of not greater than 100 kDa, the first temperature is atemperature between 0° C. and 5° C., the second temperature is atemperature between 10° C. and 40° C., and T₁ is at least 8 hours.

The methods of the invention allows manufacturing drug crystals of intight particle size distribution (PSD) ranges from very small sizes(e.g., <75 nm) to larger sizes (e.g., 5,000 nm) and allows use ofspecific sized particles, either alone, or in combination with smalleror larger sized particles of the same drug crystals made via the methodsdescribed herein, or in combination with a different form of the drug(e.g., stock material or form obtained by homogenization) or with otherexcipients (such as solvents, demulcents, mucoadehsives) to control therelease, distribution, metabolization or elimination of, or to enhancetissue penetration or tissue residence time of such drug.

In one embodiment, the drug suspension is prepared in a static batchreactor, using sonication (e.g., ultrasonication) or ultrahomogenizationto disperse the precipitating drug in the antisolvent. In oneembodiment, the ultrasonicating process is accomplished by placing in asonicating bath, providing ultrasound energy to the entire fluid. Inanother embodiment, the ultrasonicating process is accomplished using aprobe sonotrode. In yet another embodiment, the dispersion step duringprecipitation of the drug in the antisolvent, is high pressurehomogenization.

In another embodiment, the drug suspension is prepared in a flow-throughreactor, during ultrasonication or ultrahomogenization. The temperatureof the solution may be 0-4 or 2-8 degrees centigrade. In anotherembodiment, the temperature of the solution may be 22-30 degreescentigrade. The flow-through reactor may be jacketed to betemperature-controlled.

The drug solution (Phase I) is metered into the reactor by means of asyringe pump. In another embodiment, the drug suspension is metered intothe reactor by means of other automated pump devices. The flow rate ofPhase I may be in the range 0.1 ml/min to 40 ml/min. In the flow-throughreactor (or flow reactor), the flow rate of Phase I may be in the range0.1 ml/min to 40 ml/min or 0.5 to 900 ml/min (e.g., 0.5-2.0 ml/min,10-900 ml/min, 12-700 ml/min, 50-400 ml/min, 100-250 ml/min, or 110-130ml/min). In the flow-through reactor, the flow rate of Phase II may bein the range 0.1 ml/min to 40 ml/min or 2.5-2100 ml/min (e.g., 2.5-900ml/min, 2.5-2.0 ml/min, 10-900 ml/min, 12-700 ml/min, 50-400 ml/min,100-250 ml/min, or 110-130 ml/min).

Components of Phase I and Phase II in Step 1: The excipients used todissolve the drug to create the solution in Phase I are selected suchthat they are miscible and soluble in Phase II. Phase II components aresuch that this phase acts as an antisolvent only for the drug. As phaseI is added to phase II in the presence of sonication, the drugprecipitates into nanocrystals. Phase II is sterile-filtered through a0.22 micron PVDF filter into a holding container maintained at 0-4° C.or 2-8° C. Phase II is metered into a cell fitted with a sonotrode, orsonicating probe. The Phase I solution is then metered into the cellinto Phase II drop-wise, while sonicating. The nanocrystals produced byStep 1 can be held in a holding tank at 2-8° C., or 22-25° C. or 30-40°C. This process of “holding” is called annealing to stabilize thenanocrystals produced in Step 1. Annealing, or physical ageing of thenanosuspension produced in Step 1, allows the drug molecules to “relax”and arrange in its most stable thermodynamic state. The choice of theannealing temperature is dependent upon the physiochemicalcharacteristics of the drug. Time duration of annealing is alsoimportant. In one embodiment, the duration of annealing is 30 minutes.In another embodiment, the duration of annealing is between 30 minutesand 90 minutes. In another embodiment, the duration of annealing isbetween 90 minutes and 12 hours. In another embodiment, the duration ofannealing is between 12 hours and 24 hours.

The components of Phase I and Phase II are of low viscosity, so thateach phase can be sterile filtered through a 0.22 micron filter.Alternatively, the sterile filtration can be accomplished by other meansof sterilization such as autoclaving, gamma irradiation, ethylene oxide(ETO) irradiation.

The solvents to create Phase I for the initial nanosuspension may beselected from, but not limited to PEG400, PEG300, PEG100, PEG1000,PEG-Stearate, PEG40-Stearate, PEG-Laureate, lecithin, phosphatidylcholines, PEG-oleate, PEG-glycerol, TWEENs® (plysorbates), SPANs®(sorbitan monooleate), polypropylene glycol, DMSO, ethanol, isopropanol,NMP, DMF, acetone, methylene chloride, sorbitols.

The steric stabilizing solution used as Phase II for the initialnanosuspension may be selected from, but not limited to aqueoussolutions of methyl cellulose, PVP, PVA, HPMC, cellulose, PLURONIC F127®(polyoxyethylene-polyoxyproplene block copolymer), PLURONIC F68®(polyoxyethylene-polyoxyproplene block copolymer), Carbomer (acrylicacid homopolymer crosslinked with allyl sucrose or allylpentaeythritol), hydroxyethyl cellulose, hydroxypropyl cellulose, PEGs,lecithin, phosphatidyl cholines, polyquarternium-1, polylysine,polyarginine, polyhistidine, guar gums, xanthan gums, chitosans,alginates, hyaluronic acid, chondroitin sulfate, TWEEN 20®, TWEEN 80®,SPANs® (sorbitan monooleate), sorbitols, amino acids. In a preferredembodiment, the steric stabilizer is methyl cellulose of viscosity 15cP. In another embodiment, the steric stabilizer in phase II is methylcellulose of viscosity 4 cP. In another embodiment, the stericstabilizer is methyl cellulose of viscosity 50 cP. In anotherembodiment, the steric stabilizer is methyl cellulose of viscosity 4000cP. In another embodiment, the steric stabilizer is methyl cellulose ofviscosity 100,000 cP. The concentration of methyl cellulose is0.10%-0.20%, 0.20%-0.40% and 0.40%-0.50%. In a preferred embodiment, theconcentration of methyl cellulose in phase II is 0.20%. In anotherpreferred embodiment, the concentration of methyl cellulose in phase IIis 0.39%. In one embodiment, the steric stabilizer in phase II isCARBOMER 940® (acrylic acid homopolymer crosslinked with allyl sucroseor allyl pentaerythritol) in concentrations 0.1-1%, 1%-10%. In anotherembodiment, the steric stabilizer is phase II is carboxymethyl cellulosein concentrations between 0.1%-1% and 1%-10%. In another embodiment, thesteric stabilizer in phase II is carboxymethyl cellulose in combinationwith CARBOMER 940 acrylic acid homopolymer crosslinked with allylsucrose or allyl pentaerythritol). In another embodiment, the stericstabilizer in phase II is PVA in concentrations between 0.1%-1% and1-10%. In another embodiment the steric stabilizer in phase II is PVP inconcentrations between 0.1% and 10%.

The steric stabilizer can also be cationic. Examples of useful cationicsurface stabilizers include, but are not limited to, polymers,biopolymers, polysaccharides, cellulosics, alginates, phospholipids, andnonpolymeric compounds, such as zwitterionic stabilizers,poly-n-methylpyridinium, anthryul pyridinium chloride, cationicphospholipids, chitosan, polylysine, polyvinylimidazole, polybrene,polymethylmethacrylate trimethylammoniumbromide bromide (PMMTMABr),hexyldesyltrimethylammonium bromide (HDMAB),polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate,1,2 Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Amino(PolyethyleneGlycol)2000] (sodium salt) (also known as DPPE-PEG (2000)-Amine Na),Poly(2-methacryloxyethyl trimethylammonium bromide), poloxamines such asTETRONIC 908® (copolymer of poly(ethylene oxide) (PEO) andpoly(propylene oxide) (PPO), also known as POLOXAMINE 908® (copolymer ofpoly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO)), lysozyme,long-chain polymers such as alginic acid and carregenan. Other usefulcationic stabilizers include, but are not limited to, cationic lipids,sulfonium, phosphonium, and quarternary ammonium compounds, such asstearyltrimethylammonium chloride, benzyl-di(2-chloroethyl)ethylammoniumbromide, coconut trimethyl ammonium chloride or bromide, coconut methyldihydroxyethyl ammonium chloride or bromide, decyl triethyl ammoniumchloride, decyl dimethyl hydroxyethyl ammonium chloride or bromide,C₁₂-15 dimethyl hydroxyethyl ammonium chloride or bromide, coconutdimethyl hydroxyethyl ammonium chloride or bromide, myristyl trimethylammonium methyl sulphate, lauryl dimethyl benzyl ammonium chloride orbromide, lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide,N-alkyl (C₁₂₋₁₈)dimethylbenzyl ammonium chloride, N-alkyl(C₁₄₋₁₈)dimethyl-benzyl ammonium chloride, N-tetradecylidmethylbenzylammonium chloride monohydrate, dimethyl didecyl ammonium chloride,N-alkyl and (C₁₂₋₁₄) dimethyl 1-napthylmethyl ammonium chloride,trimethylammonium halide, alkyl-trimethylammonium salts anddialkyldimethylammonium salts, lauryl trimethyl ammonium chloride,ethoxylated alkyamidoalkyldialkylammonium salt and/or an ethoxylatedtrialkyl ammonium salt, dialkylbenzene dialkylammonium chloride,N-didecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl(C₁₂₋₁₄) dimethyl1-naphthylmethyl ammonium chloride and dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C₁₂, C₁₅, C₁₇ trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammoniumchloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride (ALIQUAT 336™), POLYQUAT 10™,tetrabutylammonium bromide, benzyl trimethylammonium bromide, cholineesters (such as choline esters of fatty acids), benzalkonium chloride,stearalkonium chloride compounds (such as stearyltrimonium chloride andDi-stearyldimonium chloride), cetyl pyridinium bromide or chloride,halide salts of quaternized polyoxyethylalkylamines, MIRAPOL™ andALKAQUAT™, alkyl pyridinium salts; amines, such as alkylamines,dialkylamines, alkanolamines, polyethylenepolyamines,N,N-dialkylaminoalkyl acrylates, and vinyl pyridine, amine salts, suchas lauryl amine acetate, stearyl amine acetate, alkylpyridinium salt,and alkylimidazolium salt, and amine oxides; imide azolinium salts;protonated quaternary acrylamides; methylated quaternary polymers, suchas poly[diallyl dimethylammonium chloride] and poly-[N-methyl vinylpyridinium chloride]; and cationic guar.

Components of Step 2: The components of Step 2 are selected so that thetask of purifying the nanocrystals prepared in the previous step isaccomplished. The purification process is tangential flow filtration(TFF), or normal flow filtration (NFF) to accomplish ultrafiltration, ordiafiltration, or microfiltration. In another embodiment, step 2 isaccomplished by centrifugation. The choice of the filter is dependentupon the size of the nanocrystals produced. The pore size of the filtercan be 0.1 μm, or 0.2 μm, or 0.5 μm, or 0.8 μm or 1 μm, or 10 μm, or 20μm. If the size distribution of the nanoparticles peaks at 0.5 μm, thepore size of the PVDF filter will be 0.1 μm. Preferably the size of thenanoparticles peaks at 0.5 μm. In this step, the nanocrystal suspensionis purified such the initial continuous step is replaced entirely by anew continuous phase. The new continuous phase is selected such that,the drug has minimal solubility in it. This minimizes or eliminatesOswald Ripening.

The components of the purification process may be selected from, but notlimited to the group containing aqueous solutions of HPMC, MC,carbomers, celluloses, PEGs, chitosans, alginates, PVP, F127, F68,hyaluronic acid, polyacrylic acid.

The components of Step 2 may have tissue-adhesive components that willenhance the residence time of the nanocrystals at the site, tosubsequently prolong the effectiveness of the therapy. Tissue-adhesivecomponents may be cationic or anionic. Cationic tissue-adhesivemolecules are polyquad-1, polyethyleneimine, PAMAM dendrimer, PEIdendrimer, chitosan, alginate and derivatives, thereof.

The drug nanocrystals (optionally nanosuspensions) produced by theprocesses defined can be immunomodulators to treat inflammatoryconditions of the eye. Immunomodulators have been proven effective invarious inflammatory conditions resistant to steroids, or when chronicuse of steroids is associated with steroids. Currently available agentsact as cytotoxic agents to block lymphocyte proliferation or asimmunomodulators to block synthesis of lymphokines. Cyclosporine A is apreferred immunomodulator that can be prepared using the process definedin this invention.

The drug nanosuspension can be a combination of two drugs that areformulated using the same process. Thus, it can be envisioned that bothdrugs are co-dissolved in common excipients, then precipitated using thetechniques specified in this invention.

Hydrophobic Therapeutic Agents

The term “hydrophobic therapeutic agent” or “hydrophobic drug” usedherein refers to therapeutic agents that are poorly soluble in water,e.g., having a water solubility less than about 10 mg/mL (e.g., lessthan 1 mg/mL, less than 0.1 mg/mL, or less than 0.01 mg/mL).

The methods of the invention can be applied to produce nanocrystalsand/or new morphic forms of a hydrophobic drug. Examples of hydrophobicdrugs include, but are not limited to, ROCK inhibitors, SYK-specificinhibitors, JAK-specific inhibitors, SYK/JAK or Multi-Kinase inhibitors,MTORs, STAT3 inhibitors, VEGFR/PDGFR inhibitors, c-Met inhibitors, ALKinhibitors, mTOR inhibitors, PI3K6 inhibitors, PI3K/mTOR inhibitors,p38/MAPK inhibitors, NSAIDs, steroids, antibiotics, antivirals,antifungals, antiparsitic agents, blood pressure lowering agents, cancerdrugs or anti-neoplastic agents, immunomodulatory drugs (e.g.,immunosuppressants), psychiatric medications, dermatologic drugs, lipidlowering agents, anti-depressants, anti-diabetics, anti-epileptics,anti-gout agents, anti-hypertensive agents, anti-malarials,anti-migraine agents, anti-muscarinic agents, anti-thyroid agents,anxiolytic, sedatives, hypnotics, neuroleptics, ß-blockers, cardiacinotropic agents, corticosteroids, diuretics, antiparkinsonian agents,gastro-intestinal agents, histamine H-receptor antagonists, lipidregulating agents, nitrates and other antianginal agents, nutritionalagents, opioid analgesics, sex hormones, and stimulants.

The hydrophobic drugs suitable for the methods of the invention can besteroids. Steroids include for example, fluticasone, hydrocortisone,hydrocortisone acetate, cortisone acetate, tixocortol pivalate,prednisolone, methylprednisolone, prednisone, triamcinolone acetonide,triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide,fluocinonide, fluocinolone, fluocinolone acetonide, flunisolide,fluorometholone, clobetasol propionate, loteprednol, medrysone,rimexolone, difluprednate, halcinonide, beclomethasone, betamethasone,betamethasone sodium phosphate, Ciclesonide, dexamethasone,dexamethasone sodium phosphate, dexamethasone acetate, fluocortolone,hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasonedipropionate, betamethasone valerate, betamethasone dipropionate,prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate,fluocortolone caproate, fluocortolone pivalate, fluprednidene acetate,prednisolone acetate, prednisolone sodium phosphate, fluoromethalone,fluoromethalone acetate, loteprednol etabonate, and betamethasonephosphate, including the esters and pharmaceutically acceptable saltsthereof.

The hydrophobic drugs suitable for the methods of the invention can benonsteroidal anti-inflammatory drugs, for example, Bromfenac, Diclofenacsodium, Flurbiprofen, Ketorolac tromethamine, mapracorat, naproxen,oxaprozin, ibuprofen, and nepafenac, including the esters andpharmaceutically acceptable salts thereof.

Other hydrophobic drugs suitable for the methods of the inventioninclude besifloxacin, DE-110 (Santen Inc.), Rebamipide, Androgens (DHEA,testosterone, analogs, & derivatives having poor water solubility),estrogens (poorly water soluble compounds that are derivatives ofestradiol, estriol, and estrone; e.g., estradiol, levonorgesterol,analogs, isomers or derivatives thereof), progesterone and progestins(1^(st) through 4^(th) generation) with poor water solubility (e.g.,norethindrone, analogs, and derivatives thereof, medroxyprogesterone, ortagaproget), and pregnenolone. Examples of progestins in variousgenerations include: first generation (estrane) such as norethindrone,norethynodrel, norethindrone acetate, and ethynodiol diacetate; secondgeneration (gonane) such as levonorgestrel, norethisterone, andnorgestrel; third generation (gonane) such as desogestrel, gestodene,norgestimate, and drospirenone; and fourth generation such as dienogest,drospirenone, nestorone, nomegestrol acetate and trimegestone.

Other examples of hydrophobic drugs include, e.g.,10-alkoxy-9-nitrocamptothecin, 17b-Estradiol, 3′-azido-3′-deoxythymidinepalmitate, 5-Amino levulinic acid, ABT-963, Aceclofenac, AclacinomycinA, Albendazole, Alkannin/shikonin, All-trans retinoic acid (ATRA),alpha-Tocopheryl acetate, AMG 517, amprenavir, Aprepitant, Artemisinin,Azadirachtin, Baicalein, Benzimidazole derivatives, Benzoporphyrin,Benzopyrimidine derivatives, Bicalutamide, BMS-232632, BMS-488043,Bromazepam, Bropirimine, Cabamezapine, Candesartan cilexetil,Carbamazepine, Carbendazim, Carvedilol, Cefditoren, Cefotiam,Cefpodoxime proxetil, Cefuroxime axetil, Celecoxib, Ceramide,Cilostazol, Clobetasol propionate, Clotrimazole, Coenzyme Q10, Curcumin,Cyclcoporine, Danazol, Dapsone, Dexibuprofen, Diazepam, Dipyridamole,docetaxel, Doxorubicin, Doxorubicin, Econazole, ER-34122, Esomeprazole,Etoricoxib, Etravirine, Everolimus, Exemestane, Felodipine, Fenofibrate,flurbiprofen, Flutamide, Furosemide, gamma-oryzanol, Glibenclamide,Gliclazide, Gonadorelin, Griseofulvin, Hesperetin, HO-221, Indomethacin,Insulin, Isoniazid, Isotretinoin, Itraconazole, Ketoprofen, LAB687,Limaprost, Liponavir, Loperamide, Mebendazole, Megestrol, Meloxicam,MFB-1041, Mifepristone, MK-0869, MTP-PE, Nabilone, Naringenin, Nicotine,Nilvadipine, Nimesulide, Nimodipine, Nitrendipine, Nitroglycerin,NNC-25-0926, Nobiletin, Octafluoropropane, Oridonin, Oxazepam,Oxcarbazepine, Oxybenzone, Paclitaxel, Paliperidone palmitate,Penciclovir, PG301029, PGE2, Phenytoin, Piroxicam, Podophyllotoxin,Porcine pancreatic lipase and colipase, Probucol, Pyrazinamide,Quercetin, Raloxifene, Resveratrol, Rhein, Rifampicin, Ritonavir,Rosuvastatin, Saquinavir, Silymarin, Sirolimus, Spironolactone,Stavudine, Sulfisoxazole, Tacrolimus, Tadalafil, Tanshinone, Teapolyphenol, Theophylline, Tiaprofenic acid, Tipranavir, Tolbutamide,Tolterodine tartrate, Tranilast, Tretinoin, Triamcinolone acetonide,Triptolide, Troglitazone, Valacyclovir, Verapamil, Vincristine,Vinorelbin-bitartrate, Vinpocetine, Vitamin-E, Warfarin, and XK469. Moreexamples include, e.g., amphotericin B, gentamicin and otheraminoglycoside antibiotics, ceftriaxone and other cephalosporins,tetracyclines, cyclosporin A, aloxiprin, auranofin, azapropazone,benorylate, diflunisal, etodolac, fenbufen, fenoprofen calcium,meclofenamic acid, mefanamic acid, nabumetone, oxyphenbutazone,phenylbutazone, sulindac, benznidazole, clioquinol, decoquinate,diiodohydroxyquinoline, diloxanide furoate, dinitolmide, furzolidone,metronidazole, nimorazole, nitrofurazone, ornidazole, and tinidazole.

The hydrophobic drugs suitable for the methods of the invention can alsobe FDA-approved drugs with c Log P of five or more, such as those listedin the table below.

2-(4-hydroxy-3,5-diiodobenzyl) Bromthymol blue cyclohexanecarboxylicBuclizine, buclizine hydrochloride 3,3′,4′,5-tetrachlorosalicylanilideBunamiodyl sodium 4,6-bis(1-methylpentyl)resorcinol Butenafine,butenafine hydrochloride 4,6-dichloro-2-hexylresorcinol Butoconazole,butoconazole nitrate Acitretin Calcifediol Adapalene Calcium oleateAlpha-butyl-4-hydroxy-3,5- Calcium stearate diiodohydrocinnamic acidCandesartan cilexetil Alpha-carotene Captodiame, captodiameAlpha-cyclohexyl-4-hydroxy-3,5- hydrochloride diiodohydrocinnamic acidCetyl alcohol Vitamin E Chaulmoogric acid Vitamin E acetateChloramphenicol palmitate Alverine, Alverine Citrate ChlorophenothaneAmiodarone Chlorophyll, chlorophyll unk Astemizole ChlorotrianiseneAtiprimod dihydrochloride Chlorprothixene Atorvastatin, atorvastatincalcium Cholecalciferol Benzestrol Cholesterol Bepridil, bepridilhydrochloride Choline iodide sebacate Beta-carotene CinacalcetBexarotene Cinnarizine Bithionol Dromostanolone propionate Bitolterol,bitolterol mesylate Dronabinol Clindamycin palmitate, Dutasterideclindamycin palmitate Econazole, econazole nitrate hydrochloride VitaminD2, ergocalciferol Clofazimine Ergosterol, Cloflucarban Estradiolbenzoate Clomiphene, enclomiphene, Estradiol cypionate zuclomiphene,clomiphene citrate Estradioldipropionate, estradiol Clotrimazoledipropionate Colfosceril palmitate Estradiol valerate ConivaptanEstramustine Cyverine hydrochloride, cyverine Ethanolamine oleateDesoxycorticosterone Ethopropazine, ethopropazine trimethylacetate,hydrochloride desoxycorticosterone pivalate Ethyl icosapentate,eicosapentaenoic Dextromethorphan polistirex acid ethyl ester, ethylDichlorodiphenylmethane Ethylamine oleate Diethylstilbestrol EtretinateDiethylstilbestrol dipalmitate Fenofibrate Diethylstilbestroldipropionate Fenretinide Dimestrol Flunarizine, flunarizinehydrochloride Dimyristoyl lecithin, Fluphenazine decanoateDiphenoxylate, atropine sulfate, Fluphenazine enanthate diphenoxylatehydrochloride Fosinopril, fosinopril sodium Dipipanone, dipipanoneLevomethadone hydrochloride Linoleic acid, Docosanol Lucanthone,lucanthone Docusate sodium hydrochloride Domine Meclizine, meclizineDoxercalciferol hydrochloride Fulvestrant Meclofenamic acid,meclofenamate, Gamolenic acid, gammalinolenic meclofenamate sodium acidMefenamic acid Glyceryl stearate, glyceryl Menthyl salicylatemonostearate Mercuriclinoleate Gramicidin Mercury oleate Halofantrine,halofantrine Mestilbol 5 mg, mestilbol hydrochloride Methixene,methixene hydrochloride Haloperidol decanoate Mibefradil, mibefradilHexachlorophene dihydrochloride Hexestrol Miconazole HexetidineMifepristone Humulus Mitotane Hydroxyprogesterone caproate Mometasonefuroate Hypericin Monoxychlorosene Implitapide Montelukast, montelukastsodium Indigosol Motexafin gadolinium Indocyanine green Myristyl alcoholIocarmate meglumine Nabilone Iodipamide Naftifine, naftifinehydrochloride Iodoalphionic acid Posaconazole Iodoxamate megluminePotassium oleate Iophendylate Potassium ricinoleate Isobutylsalicylcinnamate Potassium stearate Itraconazole Prednimustine Nandrolonedecanoate Probucol Nandrolone phenpropionate Progesterone caproateN-myristyl-3-hydroxybutylamine Promethestrol dipropionate hydrochloride1 mg, n myristyl 3 Pyrrobutamine phosphate Nonoxynol 9, nonoxynol,Quazepam nonoxynol 10, nonoxynol 15, Quinacrine, quinacrinehydrochloride nonoxynol 30, Quinestrol Octicizer Raloxifene, raloxifenehydrochloride Octyl methoxycinnamate Ritonavir Oleic acid Rose bengal,rose bengal sodium Omega 3 acid ethyl esters Sertaconazole OrlistatSertraline, sertraline hydrochloride Oxiconazole, oxiconazole nitrateSibutramine, sibutramine Oxychlorosene hydrochloride Pararosanilinepamoate Rapamycin, sirolimus, rapamune Penicillin v hydrabamineSitosterol, sitosterols Perflubron Sodium beta-(3,5-diiodo-4-Perhexiline, perhexiline maleate hydroxyphenyl)atropate, PermethrinSodium dodecylbenzenesulfonate ng, Vitamin K, phytonadionedodecylbenzenesulfonic acid Pimecrolimus Thymol iodide PimozideTioconazole Polyethylene, Tipranavir Polyvinyl n-octadecyl carbamateTiratricol Porfimer, porfimer sodium Tocopherols excipient Sodium oleateTolnaftate Tetradecylsulfate, sodium Tolterodine tetradecyl sulfateToremifene, toremifene citrate Sorbitan-sesquioleate Alitretinoin,isotretinoin, Stearic acid neovitamin a, retinoic acid, Sulconazole,sulconazole nitrate tretinoin, 9-cis-retinoic Suramin, suraminhexasodium Tribromsalan Tacrolimus Triolein I 125 Tamoxifen, tamoxifencitrate Triparanol Tannic acid Troglitazone Tazarotene TyloxapolTelithromycin Tyropanoate, tyropanoate sodium Telmisartan Ubidecarenone,coenzyme Q10 Temoporfin Verapamil, dexverapamil Temsirolimus,tezacitabine Verteporfin Terbinafine Vitamin A acetate TerconazoleVitamin A palmitate Terfenadine Zafirlukast Testosterone cypionate Cetylmyristate Testosterone enanthate Phenylbutazone, phenylbutazoneTestosterone phenylacetate isomer Tetradecylamine lauryl sarcosinateBryostatin-1 Thioridazine Dexanabinol Cetyl myristoleate Dha-paclitaxelDocosahexanoic acid, doconexent Disaccharide tripeptide glycerol Hemindipalmitoyl Lutein Oxiconazole nitrate Chlorophyll b from spinachSarsasapogenin Gossypol Tetraiodothyroacetic acid Imipramine pamoate(NZ)-N-[10,13-dimethyl-17-(6- Iodipamide meglumine methylheptan-2-yl)-Ondascora Zinc stearate

The hydrophobic drugs suitable for the methods of the invention can alsobe FDA-approved drugs with A Log P of five or more, such as those listedin the table below.

tocoretinate bitolterol mesilate indocyanin green, Daiichifalecalcitriol colfosceril palmitate ioxaglic acid octenidinefesoterodine fumarate gadofosveset trisodium quazepam probucolfosaprepitant dimeglumine talaporfin sodium levocabastine menatetrenoneciclesonide miriplatin hydrate mometasone furoatethiamine-cobaltichiorophyllate revaprazan montelukast sodium mometasonefuroate, nasal everolimus mometasone furoate, DPI, everolimus elutingstent Twisthaler dexamethasone linoleate mometasone furoate + formoterolestramustine phosphate sodium mometasone furoate, Almirall zotarolimustiotropium bromide + formoterol Lipo-dexamethasone palmitate fumarate +ciclesonide, Cipla temoporfin mometasone furoate, implant, artemether +lumefantrine Intersect ENT acetoxolone aluminium salt clobetasonebutyrate pipotiazine palmitate isoconazole telmisartan miconazole +benzoyl peroxide telmisartan + Hydrochlorothiazide miconazole nitrate(HCTZ) miconazole telmisartan + amlodipine, BI miconazole(S)-amlodipine + telmisartan miconazole, Barrier sirolimus miconazole,buccal, sirolimus, NanoCrystal bilastine sirolimus, stent, Cordis-1dexamethasone cipecilate temsirolimus etretinate docosanol tibenzoniumclofoctol mepitiostane iodoxamate meglumine etravirine AGP-103 synthconjugated estrogens, B itraconazole sulconazole itraconazole, Choongwaeormeloxifene itraconazole, Barrier blonanserin halofantrine eveningprimrose oil etiroxate flutrimazole testosterone undecanoate gammalinolenic acid meglumine iotroxinate SH-U-508 teboroxime lofepraminetirilazad mesylate treprostinil sodium fazadinium bromide rimexolonefospropofol disodium treprostinil sodium, inhaled amiodarone dienogest +estradiol valerate amiodarone estradiol + levonorgestrel (patch)fulvestrant xibornol indometacin farnesil sodium prasterone sulfate, S-Pmelinamide ethyl icosapentate, Amarin miltefosine bepridil candesartancilexetil bifonazole candesartan cilexetil + HCTZ lonazolac calciumcandesartan cilexetil + amlodipine amorolfine cytarabine ocfosfateterbinafine penfluridol amorolfine, nail, Kyorin paliperidone palmitatepitavastatin zuclopenthixol decanoate perflexane prednisolone farnesilalprazolam atorvastatin calcium alprazolam atorvastatin calcium +amlodipine alprazolam atorvastatin strontium sertaconazoleatorvastatin + fenofibrate telithromycin (micronized), Ethypharmzafirlukast ASA + atorvastatin + ramipril + diclofenac once-dailymetoprolol ER diclofenac potassium (S)-amlodipine + atorvastatindiclofenac sodium, Diffucaps prednimustine diclofenac twice-dailyfidaxomicin diclofenac terfenadine diclofenac, Applied-1 orlistatdiclofenac bexarotene diclofenac bexarotene, gel, Ligand rifaximincalcium carbonate + vitamin D3 rifaximine cream alendronate sodium +vitamin D diclofenac sodium omega-3-acid ethyl esters diclofenacpotassium pasireotide diclofenac sodium gel ebastine diclofenacpotassium, ophthalm ebastine, oral dissolving diclofenac potassiumenocitabine diclofenac sodium Malarex pimozide pimecrolimus nabiximolsfosamprenavir calcium dronabinol clinofibrate dronedarone hydrochloridetolciclate sestamibi teprenone acitretin dexamethasone sodium phosphatepramiverine adapalene setastine fenticonazole rilpivirine ixabepilonemifepristone Epiduo seratrodast Efalex azilsartan brotizolammifepristone eltrombopag olamine atracurium besilate bazedoxifeneacetate cisatracurium besylate butenafine eberconazole Cloderminastemizole + pseudoephedrine chlorhexidine iopromide chlorhexidineotilonium bromide estradiol valerate + norethisterone Piloplex enanthateporfimer sodium cinacalcet hydrochloride benzbromarone ethylicosapentate tamibarotene fexofenadine HCl eprosartan mesylatefexofenadine + pseudoephedrine riodoxol almitrine bismesilate eprosartanmesylate + HCTZ butoconazole ivermectin butoconazole naftifine TBI-PABquinestrol medroxyprogesterone, depot raloxifene hydrochloridemedroxyprogesterone acetate LA repaglinide dutasteride metformin +repaglinide flunarizine econazole nitrate dutasteride + tamsulosinberaprost liranaftate beraprost sodium, SR nabilone vinfluninelidoflazine ethinylestradiol + norelgestromin ethanolamine oleatedenaverine hydrochloride lasofoxifene aprepitant maraviroc fluocortinbutyl tacrolimus monosialoganglioside GM-1, Amar tacrolimus,modified-release monosialoganglioside GM1 tacrolimus, topical irbesartantacrolimus irbesartan + HCTZ Americaine amlodipine besilate +irbesartan, conivaptan hydrochloride Dainippon posaconazole tolvaptanetizolam promestriene tipranavir Epavir azulene sodium sulfonateufenamate triazolam aprindine triazolam clobenoside hydroxyprogesteronecaproate, atazanavir sulfate Hologic proglumetacin mifamurtide gemeprostlopinavir + ritonavir rifapentine ritonavir sofalcone ritonavir, softgel-2 motretinide meclofenamate sodium verapamil alfacalcidol verapamilegualen sodium verapamil, OROS tamoxifen verapamil tamoxifen verapamiltoremifene citrate verapamil SR tamoxifen, oral liquid, Savienttrandolapril + verapamil Efamol Marine verapamil terconazole verapamilhydrochloride fluvastatin valsartan fluvastatin, extended releasevalsartan + HCTZ losartan + HCTZ amlodipine + valsartan losartanpotassium enzalutamide amlodipine + losartan Sm153 lexidronam(S)-amlodipine + losartan lubiprostone beclometasone dipropionate, 3Mparicalcitol beclometasone dipropionate, LA paricalcitol, oralclotrimazole amineptine beclometasone dipropionate, Dai isopropylunoprostone beclometasone + formoterol loperamide heme arginateloperamide tolterodine promegestone tolterodine, extended-releasesertraline hydrochloride oxiconazole

Other drugs suitable for the methods of the invention include longacting bronchodilators (e.g., Salmeterol xinafoate and Formoterol),anti-inflammatory drugs (statins such as Atorvastatin, Simvastatin,Lovastatin, and Rosuvastatin), macrolide antibiotics (e.g.,Azithromycin), antinauseants, drugs highly metabolized by first passmetabolism (e.g., imipramine, morphine, buprenorphine, propranolol,diazepam, and midazolam), protein therapeutics (e.g., ranibizumab,bevacizumab, Aflibercept), rilonacept, and those listed in the tablebelow.

Exemplary Route/ Drug Name Exemplary Indications Dosage Form AzolesSeborrhea, Tinea, Tinea Topical, Ketoconazole versicolor, SkinOphthalmic Itraconazole inflammation, Athlete's formulation Fluconazolefoot, Oral candidiasis, (e.g., ophthalmic Posaconazole Histoplasmosis,Cushing's antifungal Voriconazole syndrome, Blastomycosis, formulation)Isavuconazole Coccidioidomycosis, Miconazole Paracoccidioidomycosis,Terconazole Leishmaniasis, Chronic Butoconazole mucocutaneouscandidiasis, Tioconazole Acanthamoeba keratitis, VulvovaginalCandidiasis Allylamine Fluconazole, Itraconazole, terbinafine Ketonazolehave activity against yeast keratitis and endophthalmitis EchinocandinsIndicated against Aspergillus Oral, Anidulafungin and Candida species,Topical (e.g., Anidulafungin approved for treating for esophagealcandidases Candida infections, especially for azole- resistant strains)Haloprogin Broad spectrum antifungal Tolnaftate Broad spectrumantifungal Naftifine Broad spectrum antifungal Butenafine Broad spectrumantifungal Ciclopirox Olamine Broad spectrum antifungal, e.g., C.albicans, E. floccosum, M. Canis Griseofulvin Tinea capitis, ringworm,Topical tinea pedis, nail fungus Fluticasone Psoriasis TopicalDesoximetasone Calcipotriol Betamethasone Topical dipropionateClobetasol propionate Diflorasone diacetate Halobetasol propionateAmcinonide Fluocinonide Diflorasone diacetate Halcinonide Momentasonefuroate Hydrocortizone valerate Desonide Amcinonide Fluocinoloneacetonide Cyclosporin Alopecia Areata Topical (autoimmune disorder)Atopic dermatitis Psoriasis Dry eye Latanoprost, Androgenetic AlopeciaTopical Bimatoprost, (Hair growth) Travoprost, and other Glaucomaprostaglandins or analogs thereof Minoxidil Androgenetic AlopeciaTopical (Hair growth) Tacrolimus Psoriasis Topical Dapsone Dermatitisherpetiformis Oral and Topical and leprosy; dermatosis, pustularpsoriasis Clindamycin Acne Topical Tretinoin Acne, cutaneous Kaposi'sSarcoma Systemic retinoids acne, psoriasis, ichthyosis, Oral, orEtretinate Darier's disease, rosacea formulated Bexarotene for dermalAcitretin psoriasis Oral and topical Isotretinoin Acne, ChemotherapyTopical, Systemic azelastine Allergy Nasal Beclomethasone Allergy NasalFlunisolide allergy Nasal Budesonide Nasal Imiquimod Genital warts,actinic Topical keratoses and certain types of skin cancer calledsuperficial basal cell carcinoma. Zanamivir Inhalation Camptothecinchemotherapeutic Oral Erlotinib chemotherapeutic Lapatinibchemotherapeutic Sorafenib chemotherapeutic Oral, or ophthalmicformulation against ARMD, DR Azithromycin Conjunctivitis OphthalmicBacitracin Conjunctivitis, Blepharitis, Keratitis, Corneal ulcers,natamycin antifungal approved for Ophthalmic ophthalmic Amphotericin BPotential ophthalmic-yeast Ophthalmic and fungal keratitis andendophthalmitis Psoralens and UVA Orally administered 8- Oral or topicalmethoxypsoralen + UV-A formulation of light therapy is a FDA-psoralens + approved treatment for light therapy psoriasis and vitiligo.Permethrin Insect repellent, lice Oral and topical FinasterideAndrogenetic alopecia Oral, Topical scalp therapy

Additional examples of hydrophobic drugs can also be found in e.g.,Biopharmaceutics Classification System (BCS) database by Therapeuticsystems Research Laboratory, Inc., Ann Arbor, Mich.; M Linderberg, etal., “Classification of Orally Administered Drugs on the WHO Model Listof Essential Medicines According to the Biopharmaceutics ClassificationSystem,” Eur J Pharm & Biopharm, 58:265-278(2004); N A Kasim et al.,“Molecular properties of WHO Essential Drugs & ProvisionalBiopharmaceutical Classification,” Molec Pharm, 1(1):85-96 (2004); ADahan & GL Amidon, “Provisional BCS Classification of the Leading OralDrugs on the Global Market,” in Burger's Medicinal Chemistry, DrugDiscovery & Development, 2010; Elgart A, et al. Lipospheres and pro-nanolipospheres for delivery of poorly water soluble compounds. Chem. Phys.Lipids. 2012 May; 165(4):438-53; Parhi R, et al., Preparation andcharacterization of solid lipid nanoparticles-a review. Curr Drug DiscovTechnol. 2012 March; 9(1):2-16; Linn M, et al. SOLUPLUS® (polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol graft co-polymer) asan effective absorption enhancer of poorly soluble drugs in vitro and invivo. Eur J Pharm Sci. 2012 Feb. 14; 45(3):336-43; Salnstio P J, et al.Advanced technologies for oral controlled release: cyclodextrins fororal controlled release. AAPS PharmSciTech. 2011 December;12(4):1276-92. PMCID: PMC3225529; Kawabata Y, et al. Formulation designfor poorly water-soluble drugs based on biopharmaceutics classificationsystem: basic approaches and practical applications. Int J Pharm. 2011Nov. 25; 420(1):1-10; van Hoogevest P, et al. Drug delivery strategiesfor poorly water-soluble drugs: the industrial perspective. Expert OpinDrug Deliv. 2011 November; 8(11):1481-500; Bikiaris D N. Soliddispersions, part I: recent evolutions and future opportunities inmanufacturing methods for dissolution rate enhancement of poorlywater-soluble drugs. Expert Opin Drug Deliv. 2011 November;8(11):1501-19; Singh A, et al. Oral formulation strategies to improvesolubility of poorly water-soluble drugs. Expert Opin Drug Deliv. 2011October; 8(10):1361-78; Tran PH-L, et al. Controlled release systemscontaining solid dispersions: strategies and mechanisms. Pharm Res. 2011October; 28(10):2353-78; Srinarong P, et al. Improved dissolutionbehavior of lipophilic drugs by solid dispersions: the productionprocess as starting point for formulation considerations. Expert OpinDrug Deliv. 2011 September; 8(9):1121-40; Chen H, et al. Nanonizationstrategies for poorly water-soluble drugs. Drug Discov. Today. 2011April; 16(7-8):354-60; Kleberg K, et al. Characterising the behaviour ofpoorly water soluble drugs in the intestine: application of biorelevantmedia for solubility, dissolution and transport studies. J. Pharm.Pharmacol. 2010 November; 62(11):1656-68; and He C-X, et al.Microemulsions as drug delivery systems to improve the solubility andthe bioavailability of poorly water-soluble drugs. Expert Opin DrugDeliv. 2010 April; 7(4):445-60; the contents of each of which areincorporated herein by reference in their entireties.

The nanocrystals of the hydrophobic drugs produced by the methods areideally suited for systemic or non-systemic treatment of disorders thatthe hydrophobic drugs are used for, such as inflammatory disorders,respiratory disorders, autoimmune diseases, cardiovascular diseases, andcancer. For example, the nanocrystals of the invention can be used fortreating rheumatoid arthritis, Lupus (including, e.g., Lupus nephritisand Systemic Lupus Erythematosus), allergic asthma, Lymphoma (includinge.g., Non-Hodgkin lymphoma and Chronic lymphocytic leukemia), Immunethrombocytopenic purpura, Psoriasis, Psoriatic arthritis, Dermatitis,Ankylosing spondylitis, Crohn's disease, Ulcerative colitis, Gout,Atopic dermatitis, Multiple sclerosis, Pemphigous (including Bullouspemphigoid), Autoimmune hemolytic anemia, Chronic inflammatorydemyelinating polyneuropathy, Guillain-Barre syndrome, Wegener'sgranulomatosis, and/or Glomerulonephritis. The nanocrystals of theinvention can also be used in the primary prevention of major adversecardiac events in patients with coronary artery disease.

New Morphic Forms

One unexpected advantage of the methods of the invention is that thenanocrystals of the hydrophobic drugs produced via the methods havenovel morphologies different from those of the commercially availablestock material or known morphologies of the hydrophobic drugs. The novelmorphologies can be more stable (e.g., thermally stable), having highertap densities, and/or more crystalline.

In one aspect, this invention provides a novel morphic form offluticasone propionate, i.e., Form A, which is characterized by an X-raypowder diffraction pattern including peaks at about 7.8, 15.7, 20.8,23.7, 24.5, and 32.5 degrees 2θ.

For example, Form A is further characterized by an X-ray powderdiffraction pattern including additional peaks at about 9.9, 13.0, 14.6,16.0, 16.9, 18.1, and 34.3 degrees 2θ.

For example, Form A is characterized by an X-ray powder diffractionpattern including peaks listed in Table A below.

TABLE A 2theta d value Intensity (degree) (Å) counts (I) I/I0 % I 7.77811.3667 242 0.11 2.030712 9.933 8.9044 2170 1 18.20928 11.463 7.7191 820.04 0.688093 12.34 7.1724 111 0.05 0.931442 12.998 6.8107 214 0.11.795754 14.648 6.0471 1,059 0.49 8.886465 15.699 5.6447 1,987 0.9216.67366 16.038 5.5262 385 0.18 3.230679 16.896 5.2473 985 0.45 8.26550318.101 4.9007 353 0.16 2.962155 19.342 4.5889 121 0.06 1.015356 20.0854.4209 266 0.12 2.232105 20.838 4.2627 645 0.3 5.412436 22.003 4.0396259 0.12 2.173366 22.763 3.9064 146 0.07 1.225141 23.705 3.7532 594 0.274.984476 24.52 3.6304 996 0.46 8.357808 25.621 3.4768 129 0.06 1.08248726.141 3.4088 122 0.06 1.023748 26.853 3.32 247 0.11 2.072669 32.4622.758 342 0.16 2.86985 34.293 2.6149 267 0.12 2.240497 34.736 2.5825 1950.09 1.636318

For example, Form A is characterized by nanocrystals having themorphology of a long plate or blade.

For example, Form A is substantially free of impurities.

For example, Form A has a purity of greater than 90%, greater than 92%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,or greater than 99%.

For example, Form A has a tap density of 0.5786 g/cm³. In contrast, thetap density of fluticasone propionate stock is 0.3278 g/cm³.

For example, the heat of melting for Form A is significantly higher(54.21 J/g), indicating that the former is a more crystalline material,requiring more energy to break inter-molecular bonds such as ionic andhydrogen bonds.

For example, Form A has a melting range of 10° C., also indicating ahighly ordered microstructure. In contrast, fluticasone propionate stockmaterial melts over a slight wider range (11.1° C.).

For example, Form A dissolves more slowly than the stock material orhomogenized material. Form A reaches saturated solubility after 6 weeksof incubation in an aqueous medium while the stock material orhomogenized material reaches saturated solubility within 2 weeks ofincubation in an aqueous medium.

For example, Form A is characterized by a dissolution rate in an aqueousmedium (e.g., water or an aqueous solution) of about 1 μg/g/day in waterat room temperature.

For example, the unit cell structure of Form A is Monoclinic, P21,a=7.7116 Å, b=14.170 Å, c=11.306 Å, beta=98.285, volume 1222.6.

For example, Form A has a melting point of 299.5° C., as opposed to297.3° C. for the stock material (polymorph 1).

For example, Form A is characterized by nanoplates with an average sizeof about 10-10000 nm, (e.g., 100-1000 nm or 300-600 nm).

For example, Form A is characterized by fluticasone propionatenanoplates with a narrow range of size distribution. For example, Form Ais characterized by fluticasone propionate nanoplates with a sizedistribution of 50-100 nm, of 100-300 nm, of 300-600 nm, of 400-600 nm,of 400-800 nm, of 800-2000 nm, of 1000-2000 nm, of 1000-5000 nm, of2000-5000 nm, of 2000-3000 nm, of 3000-5000 nm, or of 5000-10000 nm.

For example, the nanoplates each have a thickness between 5 nm and 200nm (e.g., 10-150 nm or 30-100 nm).

For example, the nanoplates have the [001] crystallographic axissubstantially normal to the surfaces that define the thickness of thenanoplates.

In another aspect, this invention provides a novel morphic form oftriamcinolone acetonide, i.e., Form B, which is characterized by anX-ray powder diffraction pattern including peaks at about 11.9, 13.5,14.6, 15.0, 16.0, 17.7, and 24.8 degrees 2θ.

For example, Form B is further characterized by an X-ray powderdiffraction pattern including additional peaks at about 7.5, 12.4, 13.8,17.2, 18.1, 19.9, 27.0 and 30.3 degrees 2θ.

For example, Form B is characterized by an X-ray powder diffractionpattern including peaks listed in Table B below.

TABLE B 2theta d value Intensity Relative (degree) (Å) (cps) Intensity7.5265 11.73621 925.01 1.86 11.9231 8.89089 36615.41 73.8 12.35617.82749 3250.64 6.55 13.4675 7.09394 4914.03 9.9 13.8284 6.73828 1483.262.99 14.5734 6.07325 49613.49 100 15.0476 5.88291 17123.8 34.51 15.95765.54942 10066.26 20.29 17.2466 5.13746 9609.43 19.37 17.6737 5.0142418104.74 36.49 18.0594 4.90802 9517.13 19.18 19.9414 4.44887 9426.99 1920.3221 4.36638 2783.08 5.61 21.3275 4.16275 1140.83 2.3 22.6548 3.921781719.17 3.47 22.9528 3.87154 1148.04 2.31 23.5648 3.77235 388.92 0.7824.7819 3.58977 15106.92 30.45 25.0765 3.54827 1873.17 3.78 25.62793.47315 1345.05 2.71 26.4662 3.36501 2669.5 5.38 27.0149 3.2979 6198.2712.49 28.6085 3.11772 2865.29 5.78 28.8669 3.09039 190.73 0.38 29.35383.04023 1382.62 2.79 30.0926 2.96725 1987.77 4.01 30.3395 2.943674605.47 9.28 30.5632 2.92263 1072.11 2.16 31.0498 2.87793 1892.56 3.8132.0078 2.79393 1593.63 3.21 32.2282 2.77533 1331.46 2.68 32.67462.73843 958.6 1.93 33.5827 2.66643 2812.44 5.67 33.7886 2.65064 1308.182.64 34.2731 2.61428 777.59 1.57 34.8978 2.5689 792.47 1.6 35.33322.53823 1252.96 2.53 35.7276 2.51111 517.17 1.04 36.3522 2.46939 317.670.64 36.5664 2.45541 1046.14 2.11 36.7679 2.44241 354.44 0.71 37.98562.36687 2169.29 4.37 38.5534 2.33331 175.82 0.35 39.3381 2.28855 1348.092.72 39.5372 2.27749 842.58 1.7 39.9377 2.25557 1022.85 2.06

For example, Form B is characterized by an X-ray powder diffractionpattern substantially similar to the profile in red in FIG. 39 .

For example, Form B is substantially free of impurities.

For example, Form B has a purity of greater than 90%, greater than 92%,greater than 95%, greater than 96%, greater than 97%, greater than 98%,or greater than 99%.

Pharmaceutical Compositions

The invention also features pharmaceutical compositions comprising aneffective amount of the hydrophobic drug nanocrystals described hereinand a pharmaceutically acceptable carrier useful for the systemic ornon-systemic treatment or alleviation of disorders that the hydrophobicdrug is used for, e.g., inflammatory disorders such as ophthalmicdisorders and dermatologic disorders, respiratory disorders such asasthma or COPD, or cancer such as lymphoma.

In one embodiment, the invention features novel topical pharmaceuticalcompositions comprising an effective amount of nanocrystals of ahydrophobic drug (e.g., fluticasone) and a pharmaceutically acceptablecarrier useful for the treatment or alleviation of a sign or symptom andprevention of blepharitis and or meibomian gland dysfunction (MGD). Aneffective amount of the formulations of the invention may be used todecrease inflammation of the eyelid margin, thereby treating blepharitisand or MGD.

For example, the compositions described in the invention can be used forpost-operative care after surgery. For example, the composition of theinvention can be used to control of pain after surgery, control ofinflammation after surgery, argon laser trabceuloplasty andphotorefractive procedures. Furthermore, the compositions can be used totreat other ophthalmic disorders such as ophthalmic allergies, allergicconjunctivitis, cystoid macular edema or meibomian gland dysfunction.

Additionally, the composition described in the invention can be used forthe systemic or non-systemic treatment or alleviation of a sign orsymptom and prevention of dermatologic disorders such as atopicdermatitis, dermatologic lesion, eczema, psoriasis, or rash.

Signs and symptoms that are associated with blepharitis include forexample, eyelid redness, eyelid swelling, eyelid discomfort, eyeliditching, flaking of eyelid skin, and ocular redness.

Signs and symptoms of abnormal meibomian secretions include but are notlimited to increased meibomian secretion viscosity, opacity, color, aswell as an increase in the time (refractory period) between glandsecretions. Signs and symptoms of diseases associated with abnormalmeibomian gland (e.g. MGD) secretions include but are not limited to dryeye, redness of the eyes, itching and/or irritation of the eyelidmargins and edema, foreign body sensation, and matting of the lashes

The active agent component improves treats, relieves, inhibits,prevents, or otherwise decreases the signs and symptoms of blepharitisand/or MGD. The compositions of the invention are comfortable uponapplication to the eye, eye lid, eye lashes, or eye lid margin of asubject, and may be used for relief of acute or chronic blepharitisand/or MGD, and are particularly suitable for both intermittent and longterm use.

Also, the composition described in the invention can be used for thesystemic or non-systemic treatment, alleviation of a sign or symptom andprevention of respiratory disorders (e.g., asthma or COPD), autoimmunediseases (e.g., lupus or psoriasis), and cancer (e.g., lymphoma).

Fluticasone including the esters and pharmaceutically acceptable saltsthereof. Fluticasone propionate is the preferred pharmaceuticallyacceptable salt. Fluticasone propionate, also known asS-fluoromethyl-6-α-9-difluoro-11-β-hydroxy-16-α-methyl-3-oxoandrosta-1,4-diene-17-β-carbothioate,17-propionate, is a synthetic, trifluorinated, corticosteroid having thechemical formula C₂₅H₃₁F₃O₅S. It is a white to off-white powder with amolecular weight of 500.6 g/mol. Fluticasone propionate is practicallyinsoluble in water (0.14 μg/ml), freely soluble in dimethyl sulfoxideand dimethyl-formamide, and slightly soluble in methanol and 95%ethanol.

Pharmaceutical ophthalmic formulations typically contain an effectiveamount, e.g., about 0.0001% to about 10% wt/vol., preferably about0.001% to about 5%, more preferably about 0.01% to about 3%, even morepreferably about 0.01% to about 1% of an ophthalmic drug (e.g.,fluticasone) suitable for short or long term use treating or preventingophthalmic and dermatologic disorders. The amount of the ophthalmic drug(e.g., fluticasone) will vary with the particular formulation andindicated use.

Preferably, the effective amount of nanocrystals of a hydrophobic drug(e.g., fluticasone) present in the formulations should be sufficient totreat or prevent the inflammatory disorder, respiratory disorder orcancer.

In certain embodiments, the composition described herein is aslow-release composition. In other embodiments, the compositiondescribed herein is a fast-release composition. Without wishing to bebound by the theory, the drug release rate of the compositions of theinvention can be controlled by selecting specific morphic form or sizeof the drug particles. For example, the composition can includefluticasone propionate only in the morphic form of Form A or can includea mixture of Form A and polymorph 1 and/or polymorph 2 of FP. As anotherexample, the composition can include drug nanocrystals of differentsizes and/or size dispersions, e.g., a combination of nanocrystals of300-600 nm (i.e., D10-D90) and nanocrystals of about 800-900 nm (i.e.,D10-D90).

The pharmaceutical compositions of the invention described can beadministered alone or in combination with other therapies. For example,the pharmaceutical compositions of the invention described above mayadditionally comprise other active ingredients (optionally in the formof nanocrystals via the methods of this invention), including, but notlimited to, and vasoconstrictors, antiallergenic agents, anesthetics,analgesics, dry eye agents (e.g. secretagogues, mucomimetics, polymers,lipids, antioxidants), etc., or be administered in conjunction(simultaneously or sequentially) with pharmaceutical compositionscomprising other active ingredients, including, but not limited to, andvasoconstrictors, antiallergenic agents, anesthetics, analgesics, dryeye agents (e.g. secretagogues, mucomimetics, polymers, lipids,antioxidants), etc.

Formulations

The pharmaceutical compositions of the invention can be formulated invarious dosage forms suitable for the systemic or non-systemic treatmentor alleviation of disorders that the hydrophobic drug is used for, e.g.,inflammatory disorders such as ophthalmic disorders and dermatologicdisorders, respiratory disorders such as asthma, or cancer such aslymphoma. The compositions described herein can be formulated in formssuitable for the specific route of administration, e.g. topical, oral(including, e.g., oral inhalation), intranasal, enteral or parenteral(injected into the circulatory system).

In certain embodiments, the formulation described herein is aslow-release formulation. In other embodiments, the formulationdescribed herein is a fast-release formulation.

In certain embodiments, the topical compositions according to thepresent invention are formulated as solutions, suspensions, ointments,emulsions, gels, eye drops, and other dosage forms suitable for topicalophthalmic and dermatologic administration. In other embodiments, thecompositions according to the present invention are formulated as drypowers, aerosols, solutions, suspensions, ointments, emulsions, gels andother dosage forms suitable for intranasal or oral administration.

Preferably, the topical ophthalmic composition is prepared for theadministration to the eye lid, eye lashes, eye lid margin, skin, orocular surface. In addition, modifications such as sustained-releasing,stabilizing and easy-absorbing properties and the like may be furtherapplied to such the preparations. These dosage forms are sterilized, forexample, by filtration through a microorganism separating filter, heatsterilization or the like.

Aqueous solutions are generally preferred, based on ease of formulation,as well as a patient's ability to easily administer such compositions bymeans of applying the formulation to the eye lid, eye lashes and eye lidmargin. Application may be performed with an applicator, such as thepatient's finger, a WEK-CEL®, Q-TIP®, cotton swabs, polyurethane swabs,polyester swabs, 25-3318-U swabs, 25-3318-H swabs, 25-3317-U swabs,25-803 2PD swabs, 25-806 1-PAR swabs, brushes (e.g., LATISSE®(bimatoprost ophthalmic solution) brushes) or other device capable ofdelivering the formulation to the eye lid, eye lashes or eye lid margin.

However, the compositions may also be suspensions, viscous orsemi-viscous gels, or other types of solid or semisolid compositions. Inone embodiment, the formulations (e.g., fluticasone formulations) of theinvention are aqueous formulations. The aqueous formulations of theinvention are typically more than 50%, preferably more than 75%, andmost preferably more than 90% by weight water. In another embodiment,the formulations are lyophilized formulations.

In a particular embodiment, the formulations of the invention areformulated as a suspension. Such formulations generally have a particlesize no greater than 800 nm. Additionally the suspension formulation ofthe invention may include suspending and dispersing agents to preventagglomeration of the particles.

In certain embodiments, carrier is non-aqueous. The non-aqueous carriercomprises an oil, e.g., castor oil, olive oil, peanut oil, macadamia nutoil, walnut oil, almond oil, pumpkinseed oil, cottonseed oil, sesameoil, corn oil, soybean oil, avocado oil, palm oil, coconut oil,sunflower oil, safflower oil, flaxseed oil, grapeseed oil, canola oil,low viscosity silicone oil, light mineral oil, or any combinationthereof.

In embodiments wherein the formulation is an ointment, a preferredointment base used to prepare the ophthalmic ointment of the presentinvention may be one that has been used in conventional ophthalmicointments. In particular, the base may be liquid paraffin, whitepetrolatum, purified lanolin, gelation hydrocarbon, polyethylene glycol,hydrophilic ointment base, white ointment base, absorptive ointmentbase, Macrogol (Trade Name) ointment base, simple ointment base, and thelike. For example, without limitation, an ointment formulation of theinvention contains fluticasone propionate, petrolatum and mineral oil.

In embodiments wherein the formulation is a gelement, a preferredgelement base used to prepare the ophthalmic ointment of the presentinvention may be one that has been used in conventional ophthalmicgelments such as GENTEAL GEL® (hypromellose 0.2%).

In embodiments wherein the formulation is a cream, a preferred creambase used to prepare the ophthalmic cream of the present invention maybe one that has been used in conventional ophthalmic cream. For example,without limitation, a cream formulation of the invention containsfluticasone propionate, PEG 400, an oil and a surfactant.

The topical formulation may additionally require the presence of asolubilizer, in particular if the active or the inactive ingredientstends to form a suspension or an emulsion. A solubilizer suitable for anabove concerned composition is for example selected from the groupconsisting of tyloxapol, fatty acid glycerol polyethylene glycol esters,fatty acid polyethylene glycol esters, polyethylene glycols, glycerolethers, a cyclodextrin (for example alpha-, beta- or gamma-cyclodextrin,e.g. alkylated, hydroxyalkylated, carboxyalkylated oralkyloxycarbonyl-alkylated derivatives, or mono- or diglycosyl-alpha-,beta- or gamma-cyclodextrin, mono- or dimaltosyl-alpha-, beta- orgamma-cyclodextrin or panosyl-cyclodextrin), polysorbate 20, polysorbate80 or mixtures of those compounds. A specific example of an especiallypreferred solubilizer is a reaction product of castor oil and ethyleneoxide, for example the commercial products CREMOPHOR EL® (Polyoxyl 35Hydrogenated Castor Oil) or CREMOPHOR RH40® (PEG-40 Hydrogenated CastorOil). Reaction products of castor oil and ethylene oxide have proved tobe particularly good solubilizers that are tolerated extremely well bythe eye. Another preferred solubilizer is selected from tyloxapol andfrom a cyclodextrin. The concentration used depends especially on theconcentration of the active ingredient. The amount added is typicallysufficient to solubilize the active ingredient. For example, theconcentration of the solubilizer is from 0.1 to 5000 times theconcentration of the active ingredient.

Other compounds may also be added to the formulations of the presentinvention to adjust (e.g., increase) the viscosity of the carrier.Examples of viscosity enhancing agents include, but are not limited to:polysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily; vinyl polymers; and acrylic acid polymers.

In another embodiment, the topical formulations of this invention do notinclude a preservative. Such formulations would be useful for patients,who wear contact lenses, or those who use several topical ophthalmicdrops and/or those with an already compromised ocular surface (e.g. dryeye) wherein limiting exposure to a preservative may be more desirable.

Any of a variety of carriers may be used in the formulations of thepresent invention. The viscosity of the carrier ranges from about 1 cPto about 4,000,000 cP, about 1 cP to about 3,000,000, about 1 cP toabout 2,000,000 cP, about 1 cP to about 1,000,000 cP, about 1 cP toabout 500,000 cP, about 1 cP to about 400,000 cP, about 1 cP to about300,000 cP, about 1 cP to about 200,000 cP, about 1 cP to about 100,000cP, about 1 cP to about 50,000 cP, about 1 cP to about 40,000 cP, about1 cP to about 30,000 cP, about 1 cP to about 20,000 cP, about 1 cP toabout 10,000 cP, about 50 cP to about 10,000 cP, about 50 cP to about5,000 cP, about 50 cP to about 2500 cP, about 50 cP to about 1,000 cP,about 50 cP to about 500 cP, about 50 cP to about 400 cP, about 50 cP toabout 300 cP, about 50 cP to about 200 cP, about 50 cP to about 100 cP,about 10 cP to about 1000 cP, about 10 cP to about 900 cP, about 10 cPto about 800 cP, about 10 cP to about 700 cP, about 10 cP to about 600cP, about 10 cP to about 500 cP, about 10 cP to about 400 cP, about 10cP to about 300 cP, about 10 cP to about 200 cP, or about 10 cP to about100 cP.

Viscosity may be measured at a temperature of 20° C.+/−1° C. using aBROOKFIELD® Cone and Plate Viscometer Model VDV-III Ultra⁺ with a CP40or equivalent Spindle with a shear rate of approximately22.50+/−approximately 10 (1/sec), or a BROOKFIELD® Viscometer ModelLVDV-E with a SC4-18 or equivalent Spindle with a shear rate ofapproximately 26+/−approximately 10 (1/sec). Alternatively, viscositymay be measured at 25° C.+/−1° C. using a BROOKFIELD® Cone and PlateViscometer Model VDV-III Ultra⁺ with a CP40 or equivalent Spindle with ashear rate of approximately 22.50+/−approximately 10 (1/sec), or aBROOKFIELD® Viscometer Model LVDV-E with a SC4-18 or equivalent Spindlewith a shear rate of approximately 26+/−approximately 10 (1/sec).

Other compounds may also be added to the formulations of the presentinvention to adjust (e.g., increase) the viscosity of the carrier.Examples of viscosity enhancing agents include, but are not limited to:polysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily; vinyl polymers; and acrylic acid polymers.

Crystals of the present invention (e.g., fluticasone propionatecrystals) can be coated onto or impregnated into surgical or implantabledevices. In some embodiments, coating or embedding crystals (e.g.,fluticasone propionate crystals) into a surgical or implantable deviceextends the release time of the drug while providing highly localizeddrug delivery. An advantage of this mode of administration is that moreaccurate concentrations and few side effects can be achieved. In oneembodiment, the implantable device is an ocular implantable device fordrug delivery. In other embodiments, the implantable device is areservoir implant implantable by surgical means. In another embodiment,the implantable device is biodegradable, e.g., biodegradablemicroparticles. In further embodiments, the implantable device is madeof silicon, e.g., nano-structured porous silicon. Exemplary surgicaldevices include but are not limited to stents (e.g., self-expandingstents, balloon expandable coil stents, balloon expandable tubularstents and balloon expandable hybrid stents), angioplasty balloons,catheters (e.g., microcatheters, stent delivery catheters), shunts,access instruments, guide wires, graft systems, intravascular imagingdevices, vascular closure devices, endoscopy accessories. For example, adevice used in a method or composition of the invention is ISCIENCE®device, IVEENA® device, CLEARSIDE™ device, or OCUSERT® device. Coatingonto a surgical device can be performed using standard methods known inthe art, such as those referenced in US20070048433A1, the contents ofwhich are incorporated herein by reference.

Excipients

In some embodiments, the formulations of the invention comprise one ormore pharmaceutically acceptable excipients. The term excipient as usedherein broadly refers to a biologically inactive substance used incombination with the active agents of the formulation. An excipient canbe used, for example, as a solubilizing agent, a stabilizing agent, asurfactant, a demulcent, a viscosity agent, a diluent, an inert carrier,a preservative, a binder, a disintegrant, a coating agent, a flavoringagent, or a coloring agent. Preferably, at least one excipient is chosento provide one or more beneficial physical properties to theformulation, such as increased stability and/or solubility of the activeagent(s). A “pharmaceutically acceptable” excipient is one that has beenapproved by a state or federal regulatory agency for use in animals, andpreferably for use in humans, or is listed in the U.S. Pharmacopia, theEuropean Pharmacopia or another generally recognized pharmacopia for usein animals, and preferably for use in humans.

Examples of carriers that may be used in the formulations of the presentinvention include water, mixtures of water and water-miscible solvents,such as C₁- to C₇-alkanols, vegetable oils or mineral oils comprisingfrom 0.5 to 5% non-toxic water-soluble polymers, natural products, suchas gelatin, alginates, pectins, tragacanth, karaya gum, xanthan gum,carrageenin, agar and acacia, starch derivatives, such as starch acetateand hydroxypropyl starch, and also other synthetic products, such aspolyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether,polyethylene oxide, preferably cross-linked polyacrylic acid, such asneutral CARBOPOL® (polyacrylic acid crosslinked with sucrose or allypentaerythritol), or mixtures of those polymers. The concentration ofthe carrier is, typically, from 1 to 100000 times the concentration ofthe active ingredient.

Further examples of excipients include certain inert proteins such asalbumins; hydrophilic polymers such as polyvinylpyrrolidone; amino acidssuch as aspartic acid (which may alternatively be referred to asaspartate), glutamic acid (which may alternatively be referred to asglutamate), lysine, arginine, glycine, and histidine; fatty acids andphospholipids such as alkyl sulfonates and caprylate; surfactants suchas sodium dodecyl sulphate and polysorbate; nonionic surfactants such assuch as TWEEN® (polysorbate), PLURONICS® (ethylene oxide/propylene oxideblock copolymer), or a polyethylene glycol (PEG) designated 200, 300,400, or 600; a CARBOWAX® (polyethylene glycol) designated 1000, 1500,4000, 6000, and 10000; carbohydrates such as glucose, sucrose, mannose,maltose, trehalose, and dextrins, including cyclodextrins; polyols suchas mannitol and sorbitol; chelating agents such as EDTA; andsalt-forming counter-ions such as sodium.

In a particular embodiment, the carrier is a polymeric, mucoadhesivevehicle. Examples of mucoadhesive vehicles suitable for use in themethods or formulations of the invention include but are not limited toaqueous polymeric suspensions comprising one or more polymericsuspending agents including without limitation dextrans, polyethyleneglycol, polyvinylpyrolidone, polysaccharide gels, GELRITE® (Gellan Gum),cellulosic polymers, and carboxy-containing polymer systems. In aparticular embodiment, the polymeric suspending agent comprises acrosslinked carboxy-containing polymer (e.g., polycarbophil). In anotherparticular embodiment, the polymeric suspending agent comprisespolyethylene glycol (PEG). Examples of cross-linked carboxy-containingpolymer systems suitable for use in the topical stableophthalmicformulations of the invention include but are not limited toNOVEON® AA-1 (polycarbophil), CARBOPOL® (polyacrylic acid crosslinkedwith sucrose or ally pentaerythritol), and/or DURASITE® (polycarbophil,edetate disodium, sodium chloride; INSITE VISION).

In other particular embodiments, the formulations of the inventioncomprise one or more excipients selected from among the following: atear substitute, a tonicity enhancer, a preservative, a solubilizer, aviscosity enhancing agent, a demulcent, an emulsifier, a wetting agent,a sequestering agent, and a filler. The amount and type of excipientadded is in accordance with the particular requirements of theformulation and is generally in the range of from about 0.0001% to 90%by weight.

Tear Substitutes

According to some embodiments, the formulations may include anartificial tear substitute. The term “tear substitute” or “wettingagent” refers to molecules or compositions which lubricate, “wet,”approximate the consistency of endogenous tears, aid in natural tearbuild-up, or otherwise provide temporary relief of dry eye signs orsymptoms and conditions upon ocular administration. A variety of tearsubstitutes are known in the art and include, but are not limited to:monomeric polyols, such as, glycerol, propylene glycol, and ethyleneglycol; polymeric polyols such as polyethylene glycol; cellulose esterssuch hydroxypropylmethyl cellulose, carboxymethyl cellulose sodium andhydroxy propylcellulose; dextrans such as dextran 70; water solubleproteins such as gelatin; vinyl polymers, such as polyvinyl alcohol,polyvinylpyrrolidone, and povidone; and carbomers, such as CARBOMER®934P (acrylic acid homopolymer crosslinked with allyl sucrose or allylpentaerythritol), CARBOMER® 941 (acrylic acid homopolymer crosslinkedwith allyl pentaerythritol), CARBOMER® 940 (acrylic acid homoplymercrosslinked with allyl pentaerythritol, and CARBOMER® 974P (acrylic acidhomopolymer crosslinked with allyl sucrose or allyl pentaerythritol).Many such tear substitutes are commercially available, which include,but are not limited to cellulose esters such as BION TEARS® (Dextran 700.1%; hypromellose 2910 0.3%), CELLUVISC® (sodium;2,3.4,5,6-pentahydroxyhexanal; acetate), GENTEAL® (hypromellose 0.2%),OCCUCOAT® (hydroxypropyl methylcellulose ophthalmic), REFRESH®(Carboxymethylcellulose Sodium (0.5%; Glycerin (0.9%)), SYSTANE®(2-[2-(hydroxymethoxy)ethoxy]ethanol; propane-1,2-diol), TEARGEN II®(providone), TEARS NATURALE® (Hypromellose 2910 0.3%; dextran 70 0.1%),TEARS NATURAL II® (Dextran 70 0.1%; Hypromellose 2910 0.3%), TEARSNATURALE FREE® (Dextran 70 0.1%; Hypromellose 2910 0.3%), andTHERATEARS® (Sodium Carboxymethylcellulose); and polyvinyl alcohols suchas AKWA TEARS® (polyvinylalcohol 0.14%, HYPOTEARS® (polyvinyl alcohol1.0%; polyethylene glycol 400 1.0%), MOISTURE EYES® (Glycerin (0.3%);Propylene glycol (1.0%), MURINE LUBRICATING® (Polyvinyl Alocohol (0.5%),Povidone (0.6%)), and VISINE TEARS® (Glycerin 0.2%; Hypromellose 0.2%;Polyethylene glycol 400 1%), SOOTHE® (glycerinl propylene glycol). Tearsubstitutes may also be comprised of paraffins, such as the commerciallyavailable LACRI-LUBE® (mineral oil (42.5%); white petrolatum (56.8%))ointments. Other commercially available ointments that are used as tearsubstitutes include LUBRIFRESH PM® (mineral oil (15%); petrolatum (83%),MOISTURE EYES PM® (mineral oil (20%; white petrolatum (80%)) and REFRESHPM® (mineral oil (42.5%); white petrolatum (57.3%)).

In one preferred embodiment of the invention, the tear substitutecomprises hydroxypropylmethyl cellulose (Hypromellose or HPMC).According to some embodiments, the concentration of HPMC ranges fromabout 0.1% to about 2% w/v, or any specific value within said range.According to some embodiments, the concentration of HPMC ranges fromabout 0.5% to about 1.5% w/v, or any specific value within said range.According to some embodiments, the concentration of HPMC ranges fromabout 0.1% to about 1% w/v, or any specific value within said range.According to some embodiments, the concentration of HPMC ranges fromabout 0.6% to about 1% w/v, or any specific value within said range. Ina preferred embodiments, the concentration of HPMC ranges from about0.1% to about 1.0% w/v, or any specific value within said range (i.e.,0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5%, 0.5-0.6%, 0.6-0.7%, 0.7-0.8%,0.8-0.9%, 0.9-1.0%; about 0.2%, about 0.21%, about 0.22%, about 0.23%,about 0.24%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about0.29%, about 0.30%, about 0.70%, about 0.71%, about 0.72%, about 0.73%,about 0.74%, about 0.75%, about 0.76%, about 0.77%, about 0.78%, about0.79%, about 0.80%, about 0.81%, about 0.82%, about 0.83%, about 0.84%,about 0.85%, about 0.86%, about 0.87%, about 0.88%, about 0.89%, orabout 0.90%).

For example, without limitation, a tear substitute which compriseshydroxypropyl methyl cellulose is GENTEAL® (hypromellose 0.2%)lubricating eye drops. GENTEAL® (hypromellose 0.2%)(CIBAVISION®-NOVARTIS)® is a sterile lubricant eye drop containinghydroxypropylmethyl cellulose 3 mg/g and preserved with sodiumperborate. Other examples of an HPMC-based tear are provided.

In another preferred embodiment, the tear substitute comprisescarboxymethyl cellulose sodium. For example, without limitation, thetear substitute which comprises carboxymethyl cellulose sodium isREFRESH® Tears (Carboxymethylcellulose Sodium (0.5%); Glycerin (0.9%)).REFRESH® Tears (Carboxymethylcellulose Sodium (0.5%); Glycerin (0.9%))is a lubricating formulation similar to normal tears, containing a, mildnon-sensitizing preservative, stabilised oxychloro complex (PURITE®(sodium perborate)), that ultimately changes into components of naturaltears when used.

In some embodiments, the tear substitute, or one or more componentsthereof is buffered to a pH 5.0 to 9.0, preferably pH 5.5 to 7.5, morepreferably pH 6.0 to 7.0 (or any specific value within said ranges),with a suitable salt (e.g., phosphate salts). In some embodiments, thetear substitute further comprises one or more ingredients, includingwithout limitation, glycerol, propyleneglycerol, glycine, sodium borate,magnesium chloride, and zinc chloride.

Salts, Buffers, and Preservatives

The formulations of the present invention may also containpharmaceutically acceptable salts, buffering agents, or preservatives.Examples of such salts include those prepared from the following acids:hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,salicylic, citric, boric, formic, malonic, succinic, and the like. Suchsalts can also be prepared as alkaline metal or alkaline earth salts,such as sodium, potassium or calcium salts. Examples of buffering agentsinclude phosphate, citrate, acetate, and 2-(N-morpholino)ethanesulfonicacid (MES).

The formulations of the present invention may include a buffer system.As used in this application, the terms “buffer” or “buffer system” ismeant a compound that, usually in combination with at least one othercompound, provides a buffering system in solution that exhibitsbuffering capacity, that is, the capacity to neutralize, within limits,either acids or bases (alkali) with relatively little or no change inthe original pH. According to some embodiments, the buffering componentsare present from 0.05% to 2.5% (w/v) or from 0.1% to 1.5% (w/v).

Preferred buffers include borate buffers, phosphate buffers, calciumbuffers, and combinations and mixtures thereof. Borate buffers include,for example, boric acid and its salts, for example, sodium borate orpotassium borate. Borate buffers also include compounds such aspotassium tetraborate or potassium metaborate that produce borate acidor its salt in solutions.

A phosphate buffer system preferably includes one or more monobasicphosphates, dibasic phosphates and the like. Particularly usefulphosphate buffers are those selected from phosphate salts of alkaliand/or alkaline earth metals. Examples of suitable phosphate buffersinclude one or more of sodium dibasic phosphate (Na₂HPO₄), sodiummonobasic phosphate (NaH₂PO₄) and potassium monobasic phosphate(KH₂PO₄). The phosphate buffer components frequently are used in amountsfrom 0.01% or to 0.5% (w/v), calculated as phosphate ion.

A preferred buffer system is based upon boric acid/borate, a mono and/ordibasic phosphate salt/phosphoric acid or a combined boric/phosphatebuffer system. For example a combined boric/phosphate buffer system canbe formulated from a mixture of sodium borate and phosphoric acid, orthe combination of sodium borate and the monobasic phosphate.

In a combined boric/phosphate buffer system, the solution comprisesabout 0.05 to 2.5% (w/v) of a phosphoric acid or its salt and 0.1 to5.0% (w/v) of boric acid or its salt. The phosphate buffer is used (intotal) at a concentration of 0.004 to 0.2 M (Molar), preferably 0.04 to0.1 M. The borate buffer (in total) is used at a concentration of 0.02to 0.8 M, preferably 0.07 to 0.2 M.

Other known buffer compounds can optionally be added to the lens carecompositions, for example, citrates, sodium bicarbonate, TRIS, and thelike. Other ingredients in the solution, while having other functions,may also affect the buffer capacity. For example, EDTA, often used as acomplexing agent, can have a noticeable effect on the buffer capacity ofa solution.

According to some embodiments, the pH of the aqueous ophthalmic solutionis at or near physiological pH. Preferably, the pH of the aqueousophthalmic solution is between about 5.5 to about 8.0, or any specificvalue within said range. According of some embodiments, the pH of theaqueous ophthalmic solution is between about 6.5 to 7.5, or any specificvalue within said range (e.g., 6.5., 6.6., 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,7.3, 7.4, 7.5). According to some embodiments, the pH of the aqueousophthalmic solution is about 7. The skilled artisan would recognize thatthe pH may be adjusted to a more optimal pH depending on the stabilityof the active ingredients included in the formulation. According to someembodiments, the pH is adjusted with base (e.g., 1N sodium hydroxide) oracid (e.g., 1N hydrochloric acid).

For the adjustment of the pH, preferably to a physiological pH, buffersmay especially be useful. The pH of the present solutions should bemaintained within the range of 5.5 to 8.0, more preferably about 6.0 to7.5, more preferably about 6.5 to 7.0 (or any specific value within saidranges). Suitable buffers may be added, such as boric acid, sodiumborate, potassium citrate, citric acid, sodium bicarbonate, TRIS, andvarious mixed phosphate buffers (including combinations of Na₂HPO₄,NaH₂PO₄ and KH₂PO₄) and mixtures thereof. Borate buffers are preferred.Generally, buffers will be used in amounts ranging from about 0.05 to2.5 percent by weight, and preferably, from 0.1 to 1.5 percent.

According to preferred embodiments, the formulations of the presentinvention do not contain a preservative. In certain embodiments, theophthalmic formulations additionally comprise a preservative. Apreservative may typically be selected from a quaternary ammoniumcompound such as benzalkonium chloride, benzoxonium chloride or thelike. Benzalkonium chloride is better described as: N-benzyl-N—(C₈-C₁₈alkyl)-N,N-dimethylammonium chloride. Further examples of preservativesinclude antioxidants such as vitamin A, vitamin E, vitamin C, retinylpalmitate, and selenium; the amino acids cysteine and methionine; citricacid and sodium citrate; and synthetic preservatives such as thimerosal,and alkyl parabens, including for example, methyl paraben and propylparaben. Other preservatives include octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzethonium chloride, phenol,catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, phenylmercuricnitrate, phenylmercuric acetate or phenylmercuric borate, sodiumperborate, sodium chlorite, alcohols, such as chlorobutanol, butyl orbenzyl alcohol or phenyl ethanol, guanidine derivatives, such aschlorohexidine or polyhexamethylene biguanide, sodium perborate,GERMAL®II (diazolidinyl urea), sorbic acid and stabilized oxychlorocomplexes (e.g., PURITE® (sodium perborate)). Preferred preservativesare quaternary ammonium compounds, in particular benzalkonium chlorideor its derivative such as POLYQUAD®(polyquaternium-1 0.001%; (see U.S.Pat. No. 4,407,791), alkyl-mercury salts, parabens and stabilizedoxychloro complexes (e.g., PURITE® (sodium perborate)). Whereappropriate, a sufficient amount of preservative is added to theophthalmic composition to ensure protection against secondarycontaminations during use caused by bacteria and fungi.

In particular embodiments, the formulations of the invention comprise apreservative selected from among the following: benzalkonium chloride,0.001% to 0.05%; benzethonium chloride, up to 0.02%; sorbic acid, 0.01%to 0.5%; polyhexamethylene biguanide, 0.1 ppm to 300 ppm;polyquaternium-1 (Omamer M)-0.1 ppm to 200 ppm; hypochlorite,perchlorite or chlorite compounds, 500 ppm or less, preferably between10 and 200 ppm); stabilized hydrogen peroxide solutions, a hydrogenperoxide source resulting in a weight % hydrogen peroxide of 0.0001 to0.1% along with a suitable stabilizer; alkyl esters of p-hydroxybenzoicacid and mixtures thereof, preferably methyl paraben and propyl paraben,at 0.01% to 0.5%; chlorhexidine, 0.005% to 0.01%; chlorobutanol, up to0.5%; and and stabilized oxychloro complex (PURITE® (Sodium perborate))0.001% to 0.5%.

In another embodiment, the ophthalmic formulations of this invention donot include a preservative. Such formulations would be useful forpatients who wear contact lenses, or those who use several topicalophthalmic drops and/or those with an already compromised ocular surface(e.g. dry eye) wherein limiting exposure to a preservative may be moredesirable.

Viscosity Enhancing Agents and Demulcents

In certain embodiments, viscosity enhancing agents may be added to theformulations of the invention. Examples of such agents includepolysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily, vinyl polymers, and acrylic acid polymers.

A variety of viscosity enhancing agents are known in the art andinclude, but are not limited to: polyols such as, glycerol, glycerin,polyethylene glycol 300, polyethylene glycol 400, polysorbate 80,propylene glycol, and ethylene glycol, polyvinyl alcohol, povidone, andpolyvinylpyrrolidone; cellulose derivatives such hydroxypropyl methylcellulose (also known as hypromellose and HPMC), carboxymethyl cellulosesodium, hydroxypropyl cellulose, hydroxyethyl cellulose, and methylcellulose; dextrans such as dextran 70; water soluble proteins such asgelatin; carbomers such as carbomer 934P, carbomer 941, carbomer 940 andcarbomer 974P; and gums such as HP-guar, or combinations thereof. Othercompounds may also be added to the formulations of the present inventionto increase the viscosity of the carrier. Examples of viscosityenhancing agents include, but are not limited to: polysaccharides, suchas hyaluronic acid and its salts, chondroitin sulfate and its salts,dextrans, various polymers of the cellulose family; vinyl polymers; andacrylic acid polymers. Combinations and mixtures of the above agents arealso suitable.

According to some embodiments, the concentration of viscosity enhancingagent or combination of agents ranges from about 0.5% to about 2% w/v,or any specific value within said range. According to some embodiments,the concentration of viscosity enhancing agent or combination of agentsranges from about 0.5% to about 1.5% w/v, or any specific value withinsaid range. According to some embodiments, the concentration ofviscosity enhancing agent or combination of agents ranges from about0.5% to about 1% w/v, or any specific value within said range. Accordingto some embodiments, the concentration of viscosity enhancing agent orcombination of agents ranges from about 0.6% to about 1% w/v, or anyspecific value within said range. According to some embodiments, theconcentration of viscosity enhancing agent or combination of agentsranges from about 0.7% to about 0.9% w/v, or any specific value withinsaid range (i.e., about 0.70%, about 0.71%, about 0.72%, about 0.73%,about 0.74%, about 0.75%, about 0.76%, about 0.77%, about 0.78%, about0.79%, about 0.80%, about 0.81%, about 0.82%, about 0.83%, about 0.84%,about 0.85%, about 0.86%, about 0.87%, about 0.88%, about 0.89%, orabout 0.90%).

In certain embodiments, the formulations of the invention compriseophthalmic demulcents and/or viscosity enhancing polymers selected fromone or more of the following: cellulose derivatives such ascarboxymethycellulose (0.01 to 5%) hydroxyethylcellulose (0.01% to 5%),hydroxypropyl methylcellulose or hypromellose (0.01% to 5%), andmethylcelluose (0.02% to 5%); dextran 40/70 (0.01% to 1%); gelatin(0.01% to 0.1%); polyols such as glycerin (0.01% to 5%), polyethyleneglycol 300 (0.02% to 5%), polyethylene glycol 400 (0.02% to 5%),polysorbate 80 (0.02% to 3%), propylene glycol (0.02% to 3%), polyvinylalcohol (0.02% to 5%), and povidone (0.02% to 3%); hyaluronic acid(0.01% to 2%); and chondroitin sulfate (0.01% to 2%).

In one preferred embodiment of the invention, the viscosity enhancingcomponent comprises hydroxypropyl methylcellulose (HYPROMELLOSE® orHPMC). HPMC functions to provide the desired level of viscosity and toprovide demulcent activity. According to some embodiments, theconcentration of HPMC ranges from about 0% to about 2% w/v, or anyspecific value within said range. According to some embodiments, theconcentration of HPMC ranges from about 0% to about 1.5% w/v, or anyspecific value within said range. According to some embodiments, theconcentration of HPMC ranges from about 0% to about 0.5% w/v, or anyspecific value within said range.

In another preferred embodiment, the viscosity enhancing componentcomprises carboxymethyl cellulose sodium.

The viscosity of the ophthalmic formulations of the invention may bemeasured according to standard methods known in the art, such as use ofa viscometer or rheometer. One of ordinary skill in the art willrecognize that factors such as temperature and shear rate may effectviscosity measurement. In a particular embodiment, viscosity of theophthalmic formulations of the invention is measured at 20° C.+/−1° C.using a BROOKFIELD® Cone and Plate Viscometer Model VDV-III ULTRA⁺ witha CP40 or equivalent Spindle with a shear rate of approximately apprx.22.50+/−apprx 10 (1/sec), or a BROOKFIELD® Viscometer Model LVDV-E witha SC4-18 or equivalent Spindle with a shear rate of approximately26+/−apprx 10 (1/sec)).

Tonicity Enhancers

Tonicity is adjusted if needed typically by tonicity enhancing agents.Such agents may, for example be of ionic and/or non-ionic type. Examplesof ionic tonicity enhancers are alkali metal or earth metal halides,such as, for example, CaCl₂), KBr, KCl, LiCl, Nal, NaBr or NaCl, Na₂SO₄or boric acid. Non-ionic tonicity enhancing agents are, for example,urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. Theaqueous solutions of the present invention are typically adjusted withtonicity agents to approximate the osmotic pressure of normal lachrymalfluids which is equivalent to a 0.9% solution of sodium chloride or a2.5% solution of glycerol. An osmolality of about 200 to 1000 mOsm/kg ispreferred, more preferably 200 to 500 mOsm/kg, or any specific valuewithin said ranges (e.g., 200 mOsm/kg, 210 mOsm/kg, 220 mOsm/kg, 230mOsm/kg, 240 mOsm/kg, 250 mOsm/kg, 260 mOsm/kg, 270 mOsm/kg, 280mOsm/kg, 290 mOsm/kg, 300 mOsm/kg, 310 mOsm/kg, 320 mOsm/kg, 330mOsm/kg, 340 mOsm/kg, 350 mOsm/kg, 360 mOsm/kg, 370 mOsm/kg, 380mOsm/kg, 390 mOsm/kg or 400 mOsm/kg). In a particular embodiment, theophthalmic formulations of the invention are adjusted with tonicityagents to an osmolality of ranging from about 240 to 360 mOsm/kg (e.g.,300 mOsm/kg).

The formulations of the invention of the present invention may furthercomprise a tonicity agent or combination of tonicity agents. Accordingto some embodiments, the formulations of the invention may include aneffective amount of a tonicity adjusting component. Among the suitabletonicity adjusting components that can be used are those conventionallyused in contact lens care products such as various inorganic salts.Polyols and polysaccharides can also be used to adjust tonicity. Theamount of tonicity adjusting component is effective to provide anosmolality from 200 mOsmol/kg to 1000 mOsmol/kg, or any specific valuewithin said range.

Preferably, the tonicity component comprises a physiologically balancedsalt solution that mimics the mineral composition of tears. According tosome embodiments, tonicity may adjusted by tonicity enhancing agentsthat include, for example, agents that are of the ionic and/or non-ionictype. Examples of ionic tonicity enhancers are alkali metal or earthmetal halides, such as, for example, CaCl₂), KBr, KCl, LiCl, NaI, NaBror NaCl, Na₂SO₄ or boric acid. Non-ionic tonicity enhancing agents are,for example, urea, glycerol, sorbitol, mannitol, propylene glycol, ordextrose.

According to some embodiments, the tonicity component comprises two ormore of NaCl, KCl, ZnCl₂, CaCl₂), and MgCl₂ in a ratio that provides anosmolality range as above. According to some embodiments, the osmolalityrange of the formulations of the present invention is about 100 to about1000 mOsm/kg, preferably about 500 to about 1000 mOsm/kg. According tosome embodiments, the tonicity component comprises three or more ofNaCl, KCl, ZnCl₂, CaCl₂), and MgCl₂ in a ratio that provides anosmolality range of about 100 to about 1000 mOsm/kg, preferably about500 to about 1000 mOsm/kg. According to some embodiments, the tonicitycomponent comprises four or more of NaCl, KCl, ZnCl₂, CaCl₂, and MgCl₂in a ratio that provides an osmolality range of about 100 to about 1000mOsm/kg, preferably about 500 to about 1000 mOsm/kg. According to someembodiments, the tonicity component comprises NaCl, KCl, ZnCl₂, CaCl₂,and MgCl₂ in a ratio that provides an osmolality range of about 100 toabout 1000 mOsm/kg, preferably about 500 to about 1000 mOsm/kg.

According to some embodiments, NaCl ranges from about 0.1 to about 1%w/v, preferably from about 0.2 to about 0.8% w/v, more preferably about00.39% w/v. According to some embodiments, KCl ranges from about 0.02 toabout 0.5% w/v, preferably about 0.05 to about 0.3% w/v, more preferablyabout 0.14% w/v. According to some embodiments, CaCl₂) ranges from about0.0005 to about 0.1% w/v, preferably about 0.005 to about 0.08% w/v,more preferably about 0.06% w/v. According to some embodiments, MgCl₂ranges from about 0.0005 to about 0.1% w/v, preferably about 0.005 toabout 0.08% w/v, more preferably about 0.06% W/V. According to someembodiments, ZnCl₂ ranges from about 0.0005 to about 0.1% w/v,preferably about 0.005 to about 0.08% w/v, more preferably about 0.06%W/V.

According to some embodiments, the ophthalmic formulations of thepresent invention may be adjusted with tonicity agents to approximatethe osmotic pressure of normal lachrymal fluids which is equivalent to a0.9% solution of sodium chloride or a 2.5% solution of glycerol. Anosmolality of about 225 to 400 mOsm/kg is preferred, more preferably 280to 320 mOsm.

Solubilizing Agents

The topical formulation may additionally require the presence of asolubilizer, in particular if one or more of the ingredients tend toform a suspension or an emulsion. Suitable solubilizers include, forexample, tyloxapol, fatty acid glycerol polyethylene glycol esters,fatty acid polyethylene glycol esters, polyethylene glycols, glycerolethers, a cyclodextrin (for example alpha-, beta- or gamma-cyclodextrin,e.g. alkylated, hydroxyalkylated, carboxyalkylated oralkyloxycarbonyl-alkylated derivatives, or mono- or diglycosyl-alpha-,beta- or gamma-cyclodextrin, mono- or dimaltosyl-alpha-, beta- orgamma-cyclodextrin or panosyl-cyclodextrin), polysorbate 20, polysorbate80 or mixtures of those compounds. In a preferred embodiment, thesolubilizer is a reaction product of castor oil and ethylene oxide, forexample the commercial products CREMOPHOR EL® (Polyoxyl 35 HydrogenatedCastor Oil) or CREMOPHOR RH40@(PEG-40 Hydrogenated Castor Oil). Reactionproducts of castor oil and ethylene oxide have proved to be particularlygood solubilizers that are tolerated extremely well by the eye. Inanother embodiment, the solubilizer is tyloxapol or a cyclodextrin. Theconcentration used depends especially on the concentration of the activeingredient. The amount added is typically sufficient to solubilize theactive ingredient. For example, the concentration of the solubilizer isfrom 0.1 to 5000 times the concentration of the active ingredient.

Demulcifing Agents

The demulcents used in the present invention are used in effectiveamounts (i.e. “demulcifing amounts”) for providing a demulcifing effect,i.e. sufficient to lubricating mucous membrane surfaces and to relievedryness and irritation. Examples of suitable demulcents may includepolyvinyl pyrrolidone, polyvinyl alcohol, polyethylene glycol, and othercomponents such as polyethylene oxide and polyacrylic acid, arespecifically excluded. In still other embodiments, other or additionaldemulcents may be used in combination with glycerin and propyleneglycol. For example, polyvinyl pyrrolidone, polyvinyl alcohol, may alsobe used.

The specific quantities of demulcents used in the present invention willvary depending upon the application; however, typically ranges ofseveral demulcents are provided: glycerin: from about 0.2 to about 1.5%,but preferably about 1% (w/w); propylene glycol: from about 0.2 to about1.5%, but preferably about 1% (w/w); cellulose derivative: from about0.2 to about 3%, but preferably about 0.5% (w/w). If additionaldemulcents are used, they are typically used in quantities specified inthe over-the-counter monograph, cited above. A preferred cellulosederivative is pharmaceutical grade hydroxypropyl methylcellulose (HPMC).

Stability

The formulations of the present invention provide for the chemicalstability of the formulated hydrophobic drug (e.g., steroid) and otheroptional active agents of the formulation. “Stability” and “stable” inthis context refers to the resistance of the hydrophobic drug (e.g.,steroid) and other optional active agents to chemical degradation andphysical changes such as settling or precipitation under givenmanufacturing, preparation, transportation and storage conditions. The“stable” formulations of the invention also preferably retain at least90%, 95%, 98%, 99%, or 99.5% of a starting or reference amount undergiven manufacturing, preparation, transportation, and/or storageconditions. The amount of hydrophobic drug (e.g., steroid) and otheroptional active agents can be determined using any art-recognizedmethod, for example, as UV-Vis spectrophotometry and high pressureliquid chromatography (HPLC).

In certain embodiments, the formulations are stable at temperaturesranging from about 20 to 30° C. for at least 1 week, at least 2 weeks,at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks,or at least 7 weeks. In other embodiments, the formulations are stableat temperatures ranging from about 20 to 30° C. for at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 5 months,at least 6 months, at least 7 months, at least 8 months, at least 9months, at least 10 months, at least 11 months, or at least 12 months.In one embodiment, the formulation is stable for at least 3 months at20-25° C.

In other embodiments, the formulations are stable at temperaturesranging from about 2 to 8° C. for at least 1 month, at least 2 months,at least 4 months, at least 6 months, at least 8 months, at least 10months, at least 12 months, at least 14 months, at least 16 months, atleast 18 months, at least 20 months, at least 22 months, or at least 24months. In one embodiment, the formulation is stable for at least 2months at 2 to 8° C.

In other embodiments, the formulations are stable at temperatures ofabout −20° C. for at least 1 month, at least 2 months, at least 4months, at least 6 months, at least 8 months, at least 10 months, atleast 12 months, at least 14 months, at least 16 months, at least 18months, at least 20 months, at least 22 months, or at least 24 months.In one embodiment, the formulation is stable for at least 6-12 months at−20° C.

In a particular embodiment, a hydrophobic drug formulation of theinvention is stable at temperatures of about 20-30° C. at concentrationsup to 0.10% for at least 3 months. In another embodiment, theformulation is stable at temperatures from about 2-8° C. atconcentrations up to 0.10% for at least 6 months.

In some embodiments, the formulation is a sterile topical nanocrystalfluticasone propionate formulation containing a suspension of between0.001%-5% FP nanocrystals of the invention (e.g., 0.01-1%, or about0.25%, 0.1%, or 0.05%), and a pharmaceutically acceptable aqueousexcipient.

In some embodiments, the formulation further contains about 0.002-0.01%(e.g. 50 ppm±15%) benzalkonium chloride (BKC).

In some embodiments, the formulation further contains one or morecoating dispersants (e.g., Tyloxapol, polysorbate 80, and PEG stearatesuch as PEG40 stearate), one or more tissue wetting agents (e.g.,glycerin), one or more polymeric stabilizers (e.g., methyl cellulose4000 cP), one or more buffering agents (e.g., dibasic sodium phosphateNa₂HPO₄ and monobasic sodium phosphate NaH₂PO₄, and/or one or moretonicity adjusting agents (e.g., sodium chloride).

In one embodiment, the formulation includes between 0.01%-1% FPnanocrystals of the invention (e.g., about 0.25%, 0.1%, or 0.05%),benzalkonium chloride (e.g., 0.002-0.01% or about 0.005%), polysorbate80 (e.g., 0.01-1%, or about 0.2%), PEG40 stearate (e.g., 0.01-1%, orabout 0.2%), Glycerin (e.g., 0.1-10%, or about 1%), methyl cellulose4000 cP (e.g., 0.05-5%, or 0.5%), sodium chloride (e.g., 0.05-5%, or0.5%), dibasic sodium phosphate Na₂HPO₄ and monobasic sodium phosphateNaH₂PO₄, and water, and the formulation has a pH of about 6.8-7.2. Inanother embodiment, the formulation includes between 0.01%-1% FPnanocrystals of the invention (e.g., about 0.25%, 0.1%, or 0.05%),benzalkonium chloride (e.g., 0.002-0.01% or about 0.005%), Tyloxapol(e.g., 0.01-1%, or about 0.2%), Glycerin (e.g., 0.1-10%, or about 1%),methyl cellulose 4000 cP (e.g., 0.05-5%, or 0.5%), sodium chloride(e.g., 0.05-5%, or 0.5%), dibasic sodium phosphate Na₂HPO₄ and monobasicsodium phosphate NaH₂PO₄, and water, and the formulation has a pH ofabout 6.8-7.2.

In some embodiments, the formulation has a viscosity between 40-50 cP at20° C. In some embodiments, the osmolality of the formulation is about280-350 (e.g., about 285-305) mOsm/kg. In some embodiments, the pH ofthe formulation is about 6.8-7.2. In some embodiments, the formulationhas a viscosity between 40-50 cP at 20° C.

In some embodiments, the FP nanocrystals in the formulation have amedian size of 300-600 nm, a mean size of 500-700 nm, a D50 value of300-600 nm, and/or a D90 value of less than 2 μm.

In some embodiments, the formulation is administered at atherapeutically effective amount for treating blepharitis, via e.g., anapplicator (e.g., a brush such as LATISSE® (bimatoprost ophthalmicsolution) brush or a swab such as 25-3317-U swab). In one embodiment,two drops (about 40 μL drop size) of the formulation are loaded onto anapplicator (e.g., a brush or a swab) and then delivered to the subjectin need thereof by, e.g., swiping the applicator against the lowereyelid (once or twice) and then the upper eyelid (once or twice), and ifneeded, the above steps are repeated for the other eye with a newapplicator.

Methods of Use

The invention also provides the use of the formulations described hereinfor systemic or non-systemic treatment, prevention or alleviation of asymptom of a disorder the hydrophobic drug is used for, e.g.,inflammatory disorders, respiratory disorders, autoimmune diseases orcancer.

In embodiments, depending on the mode of administration, fluticasonepropionate can be used to treat, for example, respiratory relatedillnesses such as asthma, emphysema, respiratory distress syndrome,chronic obstructive pulmonary disease (COPD), chronic bronchitis, cysticfibrosis, acquired immune deficiency syndrome, including AIDS relatedpneumonia, seasonal or perennial rhinitis, seasonal or perennialallergic and nonallergic (vasomotor) rhinitis, or skin conditionstreatable with topical corticosteroids. Like other topicalcorticosteroids, fluticasone propionate has anti-inflammatory,antipruritic, and vasoconstrictive properties.

When administered in an aerosol, fluticasone propionate acts locally inthe lung; therefore, plasma levels do not predict therapeutic effect.Studies using oral dosing of labeled and unlabeled conventionalfluticasone propionate have demonstrated that the oral systemicbioavailability of fluticasone propionate is negligible (<1%), primarilydue to incomplete absorption and presystemic metabolism in the gut andliver.

The extent of percutaneous absorption of topical corticosteroids isdetermined by many factors, including the vehicle and the integrity ofthe epidermal barrier. Occlusive dressing enhances penetration. Topicalcorticosteroids can be absorbed from normal intact skin. Inflammationand/or other disease processes in the skin increase percutaneousabsorption.

Routes of Delivery

In certain embodiments, the methods of treatment disclosed in thepresent invention include all local (non-systemic) routes of delivery tothe ocular tissues and adnexa. This includes but is not limited totopical formulations such as eye drops, gels or ointments and anyintraocular, intravitreal, subretinal, intracapsular, suprachoroidal,subtenon, subconjunctival, intracameral, intrapalpebral, cul-de-sacretrobulbar and peribulbar injections or implantable or surgicaldevices.

Fluticasone propionate has been obtained in a crystalline form,designated Form 1, by dissolving the crude product (obtained, e.g. asdescribed in British Patent No. 2088877) in ethyl acetate and thenrecrystallizing. Standard spray-drying techniques have also been shownto lead only to the known Form 1 of fluticasone propionate. See U.S.Pat. No. 6,406,718 to Cooper et al. A second polymorphic form offluticasone propionate, prepared using supercritical fluid technology isdescribed in Cooper et al.

Cooper et al. describe a method for forming a particulate fluticasonepropionate product comprising the co-introduction of a supercriticalfluid and a vehicle containing at least fluticasone propionate insolution or suspension into a particle formation vessel, the temperatureand pressure in which are controlled, such that dispersion andextraction of the vehicle occur substantially simultaneously by theaction of the supercritical fluid. Chemicals described as being usefulas supercritical fluids include carbon dioxide, nitrous oxide, sulphurhexafluoride, xenon, ethylene, chlorotrifluoromethane, ethane, andtrifluoromethane. The supercritical fluid may optionally contain one ormore modifiers, such as methanol, ethanol, ethyl acetate, acetone,acetonitrile or any mixture thereof. A supercritical fluid modifier (orco-solvent) is a chemical which, when-added to a supercritical fluid,changes the intrinsic properties of the supercritical fluid in or aroundthe critical point. According to Cooper et al., the fluticasonepropionate particles produced using supercritical fluids have a particlesize range of 1 to 10 microns, preferably 1 to 5 microns.

There are several disadvantages associated with the fluticasonecompositions of Cooper et al. First, particle sizes of less than 1micron are desirable, as smaller particle sizes can be associated with amore rapid dissolution upon administration, and consequent faster onsetof action as well as greater bioavailability. Moreover, very smallfluticasone particles, i.e., less than about 150 nm in diameter, aredesirable as such compositions can be sterile filtered. In addition, thefluticasone particles of Cooper et al. may comprise supercritical fluidresidues, which are undesirable as they do not have pharmaceuticalproperties and they can potentially cause adverse reactions.

Fluticasone propionate is marketed in several different commercialforms. ADVAIR DISKUS® (GLAXOSMITHKLINE®, Research Triangle Park, N.C.)is an inhalation powder of a combination of microfine fluticasonepropionate and salmeterol xinofoate, which is a highly selective beta2-adrenergic bronchodilator. The dosage form is marketed in three dosesof fluticasone propionate: 100 mcg, 250 mcg, and 500 mcg. Followingadministration of ADVAIR® DISKUS® to healthy subjects, peak plasmaconcentrations of fluticasone propionate were achieved in 1 to 2 hours.See Physicians' Desk Reference, 57^(th) Edition, pp. 1433 (Thompson PDR,N.J. 2003). Upon administration of ADVAIR® DISKUS® 500/50 (containing500 mcg fluticasone propionate and 50 mcg salmeterol xinofoate),fluticasone propionate powder 500 mcg and salmeterol powder 50 mcg givenconcurrently, or fluticasone propionate powder 500 mcg alone, mean peaksteady-state plasma concentrations of fluticasone propionate averaged57, 73, and 70 μg/mL, respectively. Id. Peak steady-state fluticasonepropionate plasma concentration in adult patients (n=11) ranged fromundetectable to 266 μg/mL after a 500-mcg twice-daily dose offluticasone propionate inhalation powder using the DISKUS® device. Themean fluticasone propionate plasma concentration was 110 μg/mL. Thesystemic bioavailability of fluticasone propionate inhalation powderusing the DISKUS® device in healthy volunteers averages 18%. ADVAIRDISKUS® is indicated for the long-term, twice-daily, maintenancetreatment of asthma.

FLOVENT® DISKUS® (GLAXOSMITHKLINE®) is an oral inhalation powder ofmicrofine fluticasone propionate (50 mcg, 100 mcg, and 250 mcg) inlactose. Under standardized in vitro test conditions, FLOVENT® DISKUS®delivers 47, 94, or 235 mcg of fluticasone propionate from FLOVENT®DISKUS® 50 mcg, 100 mcg, and 250 mcg, respectively. The systemicbioavailability of fluticasone propionate from the DISKUS® device inhealthy adult volunteers averages about 18%. FLOVENT® DISKUS® isindicated for the maintenance treatment of asthma as prophylactictherapy, and for patients requiring oral corticosteroid therapy forasthma.

FLOVENT® ROTADISK® (GLAXOSMITHKLINE®) is an oral inhalation powder ofmicrofine fluticasone propionate (50 mcg, 100 mcg, and 250 mcg) blendedwith lactose. Under standardized in vitro test conditions, FLOVENT®ROTADISK® delivers 44, 88, or 220 mcg of fluticasone propionate fromFLOVENT® ROTADISK® 50 mcg, 100 mcg, or 250 mcg, respectively. Id. Thesystemic bioavailability of fluticasone propionate from the ROTADISK®device in healthy adult volunteers averages about 13.5%. Id. FLOVENT®ROTADISK® is indicated for the maintenance treatment of asthma asprophylactic therapy, and for patients requiring oral corticosteroidtherapy for asthma.

FLOVENT® (GLAXOSMITHKLINE®) is an oral inhalation aerosol of amicrocrystalline suspension of fluticasone propionate (44 mcg, 110 mcg,or 220 mcg) in a mixture of two chlorofluorocarbon propellants(trichlorofluoromethane and dichlorodifluoromethane) with lecithin. Eachactuation of the inhaler delivers 50, 125, or 250 mcg of fluticasonepropionate from the valve, and 44, 110, or 220 mcg, respectively, offluticasone propionate from the actuator. The systemic bioavailabilityof fluticasone propionate inhalation aerosol in healthy volunteersaverages about 30% of the dose delivered from the actuator. Peak plasmaconcentrations after an 880-mcg inhaled dose ranged from 0.1 to 1.0ng/ml. Id. FLOVENT® is indicated for the maintenance treatment of asthmaas prophylactic therapy.

FLONASE® (GLAXOSMITHKLINE®) is a nasal spray of an aqueous suspension ofmicrofine fluticasone propionate (50 mcg/dose) administered by means ofa metering, atomizing spray pump. The dosage form also containsmicrocrystalline cellulose, carboxymethylcellulose sodium, dextrose,0.02% w/w benzalkonium chloride, polysorbate 80, and 0.25% w/wphenylethyl alcohol. Indirect calculations indicate that fluticasonepropionate delivered by the intranasal route has an absolutebioavailability averaging less than 2%. After intranasal treatment ofpatients with allergic rhinitis for 3 weeks, fluticasone propionateplasma concentrations were above the level of detection (50 pg/mL) onlywhen recommended doses were exceeded and then only in occasional samplesat low plasma levels. Due to the low bioavailability by the intranasalroute, the majority of the pharmacokinetic data was obtained via otherroutes of administration. Studies using oral dosing of radiolabeled drughave demonstrated that fluticasone propionate is highly extracted fromplasma and absorption is low. Oral bioavailability is negligible, andthe majority of the circulating radioactivity is due to an inactivemetabolite. Studies comparing the effect of oral and nasal dosingdemonstrate that the therapeutic effect of FLONASE® (fluticasonepropionate (50 mcg/dose)) can be attributed to the topical effects offluticasone propionate applied to the nasal mucosa. FLONASE®(fluticasone propionate (50 mcg/dose)) nasal spray is indicated for themanagement of the nasal symptoms of seasonal and perennial allergic andnonallergic rhinitis.

CUTIVATE® (GLAXOSMITHKLINE®) is a topical dermatological fluticasonepropionate cream or ointment (0.05% and 0.005% concentration). The creamand ointment are a medium potency corticosteroid indicated for therelief of the inflammatory and pruritic manifestations ofcorticosteroid-responsive dermatoses. In a human study of 12 healthymales receiving 12.5 g of 0.05% fluticasone propionate cream twice dailyfor 3 weeks, plasma levels were generally below the level ofquantification (0.05 ng/ml). In another study of 6 healthy malesadministered 25 g of 0.05% fluticasone propionate cream under occlusionfor 5 days, plasma levels of fluticasone ranged from 0.07 to 0.39 ng/ml.In a study of 6 healthy volunteers applying 26 g of fluticasonepropionate ointment 0.005% twice daily to the trunk and legs for up to 5days under occlusion, plasma levels of fluticasone ranged from 0.08 to0.22 ng/mL.

The invention features methods of treating, preventing or alleviating asymptom of an ocular disorder such as blepharitis and/or MGD in asubject comprising use of the novel formulations described above. Forexample, a method of treating or preventing the ocular disorder (e.g.,blepharitis or MGD) may comprise administering to the eye, eye lid, eyelashes, or eye lid margin of a subject in need thereof a formulationcomprising a of the novel formulations described above.

The invention further features methods of treating dermatologicdisorders in a subject comprising use of the novel formulationsdescribed herein.

The invention further features methods of treating a respiratory disease(e.g., asthma or COPD), rhinitis, dermatitis, or esophagitis byadministering to a subject in need thereof the formulations of describedherein.

The invention also features methods of treating cancer (e.g., lymphoma)by administering to a subject in need thereof the formulations ofdescribed herein.

The invention also features methods of treating an autoimmune disease(e.g., lupus or psoriasis) by administering to a subject in need thereofthe formulations of described herein.

The effective amount of active agent to include in a given formulation,and the efficacy of a formulation for treating, preventing oralleviating a symptom of the target disorder, e.g., blepharitis and/orMGD, may be assessed by one or more of the following: slit lampevaluation, fluorescein staining, tear film breakup time, and evaluatingmeibomian gland secretions quality (by evaluating one or more ofsecretion viscosity, secretion color, gland alignment, vascularitypattern, vascularity redness, hyperkeratinization, posterior lid edge,lash, mucocutaneous junction, perigland redness, gland geometry andgland height).

The effective amount of active agent(s) in the formulation will dependon absorption, inactivation, and excretion rates of the drug as well asthe delivery rate of the active agent(s) from the formulation. It is tobe noted that dosage values may also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtimeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions. Typically, dosing will be determined using techniquesknown to one skilled in the art.

The dosage of any compound of the present invention will vary dependingon the symptoms, age and other physical characteristics of the patient,the nature and severity of the disorder to be treated or prevented, thedegree of comfort desired, the route of administration, and the form ofthe supplement. Any of the subject formulations may be administered in asingle dose or in divided doses. Dosages for the formulations of thepresent invention may be readily determined by techniques known to thoseof skill in the art or as taught herein. In embodiments, for treatingblepharitis, about 1-100 μg (e.g., 10-100 μg) FP nanoparticles areadministered to each eyelid. In one embodiment, two drops (with a totalvolume of about 80 μL) of a formulation containing FP nanocrystals(e.g., 0.01-1%, or about 0.25%, 0.1%, or about 0.05%) are applied toeach eye. For example, the two drops of formulation are first loadedonto an applicator (e.g., a brush or a swab) and then delivered to thesubject in need thereof by, e.g., swiping the applicator against thelower eyelid (once or twice) and then the upper eyelid (once or twice),and if needed, the above steps are repeated for the other eye with a newapplicator.

An effective dose or amount, and any possible effects on the timing ofadministration of the formulation, may need to be identified for anyparticular formulation of the present invention. This may beaccomplished by routine experiment as described herein. Theeffectiveness of any formulation and method of treatment or preventionmay be assessed by administering the formulation and assessing theeffect of the administration by measuring one or more indices associatedwith the efficacy of the composition and with the degree of comfort tothe patient, as described herein, and comparing the post-treatmentvalues of these indices to the values of the same indices prior totreatment or by comparing the post-treatment values of these indices tothe values of the same indices using a different formulation.

The precise time of administration and amount of any particularformulation that will yield the most effective treatment in a givenpatient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, and the like. The guidelines presented hereinmay be used to optimize the treatment, e.g., determining the optimumtime and/or amount of administration, which will require no more thanroutine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The combined use of several active agents formulated into thecompositions of the present invention may reduce the required dosage forany individual component because the onset and duration of effect of thedifferent components may be complimentary. In such combined therapy, thedifferent active agents may be delivered together or separately, andsimultaneously or at different times within the day.

Packaging

The formulations of the present invention may be packaged as either asingle dose product or a multi-dose product. The single dose product issterile prior to opening of the package and all of the composition inthe package is intended to be consumed in a single application to one orboth eyes of a patient. The use of an antimicrobial preservative tomaintain the sterility of the composition after the package is opened isgenerally unnecessary. The formulations, if an ointment formulation, maybe packaged as appropriate for an ointment, as is known to one of skillin the art.

Multi-dose products are also sterile prior to opening of the package.However, because the container for the composition may be opened manytimes before all of the composition in the container is consumed, themulti-dose products must have sufficient antimicrobial activity toensure that the compositions will not become contaminated by microbes asa result of the repeated opening and handling of the container. Thelevel of antimicrobial activity required for this purpose is well knownto those skilled in the art, and is specified in official publications,such as the United States Pharmacopoeia (“USP”) and other publicationsby the Food and Drug Administration, and corresponding publications inother countries. Detailed descriptions of the specifications forpreservation of ophthalmic pharmaceutical products against microbialcontamination and the procedures for evaluating the preservativeefficacy of specific formulations are provided in those publications. Inthe United States, preservative efficacy standards are generallyreferred to as the “USP PET” requirements. (The acronym “PET” stands for“preservative efficacy testing.”)

The use of a single dose packaging arrangement eliminates the need foran antimicrobial preservative in the compositions, which is asignificant advantage from a medical perspective, because conventionalantimicrobial agents utilized to preserve ophthalmic compositions (e.g.,benzalkonium chloride) may cause ocular irritation, particularly inpatients suffering from dry eye conditions or pre-existing ocularirritation. However, the single dose packaging arrangements currentlyavailable, such as small volume plastic vials prepared by means of aprocess known as “form, fill and seal”, have several disadvantages formanufacturers and consumers. The principal disadvantages of the singledose packaging systems are the much larger quantities of packagingmaterials required, which is both wasteful and costly, and theinconvenience for the consumer. Also, there is a risk that consumerswill not discard the single dose containers following application of oneor two drops to the eyes, as they are instructed to do, but instead willsave the opened container and any composition remaining therein forlater use. This improper use of single dose products creates a risk ofmicrobial contamination of the single dose product and an associatedrisk of ocular infection if a contaminated composition is applied to theeyes.

While the formulations of this invention are preferably formulated as“ready for use” aqueous solutions, alternative formulations arecontemplated within the scope of this invention. Thus, for example, theactive ingredients, surfactants, salts, chelating agents, or othercomponents of the ophthalmic solution, or mixtures thereof, can belyophilized or otherwise provided as a dried powder or tablet ready fordissolution (e.g., in deionized, or distilled) water. Because of theself-preserving nature of the solution, sterile water is not required.

Ophthalmic ointments may be produced as follows: if necessary,antiseptics, surfactants, stabilizers, alcohols, esters or oils areblended with an ointment base such as liquid paraffin or whitepetrolatum placed in a mortar or a mixing machine for ointment to form amixture. The ointment thus prepared is filled into a bottle or tube forointment.

Kits

In still another embodiment, this invention provides kits for thepackaging and/or storage and/or use of the formulations describedherein, as well as kits for the practice of the methods describedherein. Thus, for example, kits may comprise one or more containerscontaining one or more ophthalmic solutions, ointments suspensions orformulations, tablets, or capsules of this invention. The kits can bedesigned to facilitate one or more aspects of shipping, use, andstorage.

The kits may also optionally include a topical applicator to facilitateadministration of the formulations provided therein. In some aspects theformulations are pre-loaded in the topical applicator. Topicalapplicators include for example a swab or wand.

The kits may optionally include instructional materials containingdirections (i.e., protocols) disclosing means of use of the formulationsprovided therein. The kits may also optionally include a topicalapplicator to facilitate administration of the formulations providedtherein. While the instructional materials typically comprise written orprinted materials they are not limited to such. Any medium capable ofstoring such instructions and communicating them to an end user iscontemplated by this invention. Such media include, but are not limitedto electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g. CD ROM), and the like. Such media mayinclude addresses to internet sites that provide such instructionalmaterials.

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control. All percentages and ratios usedherein, unless otherwise indicated, are by weight. All averages usedherein, unless otherwise indicated, are number averages. For example,the average sizes of nanocrystals described herein are number averagesizes. Further, the molecular weights of polymers described herein,unless otherwise indicated, are number average molar mass of saidpolymer. As used herein, the ranges/distributions of particle size orthickness of the nanoparticles, except for the range of average sizes ofnanoparticles, are the ranges defined by D10 and D90 values.

Definitions

The term “D10” or “D10 value” refers to the value where 10% of thepopulation lies below this value. Similarly, “D90” or “D90 value” refersto the value where 90 percent of the population lies below the D90, and“D50” or “D50 value” refers to the value where 50 percent of thepopulation lies below the D50.

The term “statistical mode” or “mode” refers to the value that appearsmost often in a set of data. It is not uncommon for a dataset to havemore than one mode. A distribution with two modes is called bimodal. Adistribution with three modes is called trimodal. The mode of adistribution with a continuous random variable is the maximum value ofthe function. As with discrete distributions, there may be more than onemode.

The term “median” or “statistical median” is the numerical valueseparating the higher half of a data sample, a population, or aprobability distribution, from the lower half.

The term “abnormal meibomian gland secretion” refers to a meibomiangland secretion with increased viscosity, opacity, color and/or anincreased time (refractory period) between gland secretions.

The term “aqueous” typically denotes an aqueous composition wherein thecarrier is to an extent of >50%, more preferably >75% and inparticular >90% by weight water.

The term “blepharitis” refers to a disorder comprising inflammation ofthe eyelid in which inflammation results in eyelid redness, eyelidswelling, eyelid discomfort, eyelid itching, flaking of eyelid skin, andocular redness. Abnormal meibomian gland secretions plays a role and lidkeratinization, lid margin rounding, obscuration of the grey line,increased lid margin transparency, and increased vascularity areobserved. Although the terms meibomian gland dysfunction (MGD) andmeibomianitis are commonly referred to as blepharitis by mostinvestigators, it is important to note that these are distinct diseasesassociated with abnormal meibum (i.e., meibomian gland secretions) andthat the terms are not interchangeable. Blepharitis may cause chronicmeibomian gland dysfunction. MGD in turn will cause dry eye symptoms dueto the poor quality if the meibum which serves as the outermost layer ofthe tear film and acts to retard tear evaporation.

The term “comfortable” as used herein refers to a sensation of physicalwell being or relief, in contrast to the physical sensation of pain,burning, stinging, itching, irritation, or other symptoms associatedwith physical discomfort.

The term “comfortable ophthalmic formulation” as used herein refers toan ophthalmic formulation which provides physical relief from signs orsymptoms associated with lid margin inflammation and/or oculardiscomfort, and only causes an acceptable level of pain, burning,stinging, itching, irritation, or other symptoms associated with oculardiscomfort, when instilled in the eye.

The phrase “effective amount” is an art-recognized term, and refers toan amount of an agent that, when incorporated into a pharmaceuticalcomposition of the present invention, produces some desired effect at areasonable benefit/risk ratio applicable to any medical treatment. Incertain embodiments, the term refers to that amount necessary orsufficient to eliminate, reduce or maintain (e.g., prevent the spreadof) a symptom of eyelid margin irritation, or prevent or treat eyelidmargin inflammation. The effective amount may vary depending on suchfactors as the disease or condition being treated, the particularcomposition being administered, or the severity of the disease orcondition. One of skill in the art may empirically determine theeffective amount of a particular agent without necessitating undueexperimentation.

The phrase “pharmaceutically acceptable” is art-recognized and refers tocompositions, polymers and other materials and/or salts thereof and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andrefers to, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid (aqueous or non-aqueous) orsolid filler, diluent, excipient, solvent or encapsulating material,involved in carrying or transporting any supplement or composition, orcomponent thereof, from one organ, or portion of the body, to anotherorgan, or portion of the body, or to deliver an agent to the surface ofthe eye. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the composition and notinjurious to the patient. In certain embodiments, a pharmaceuticallyacceptable carrier is non-pyrogenic. Some examples of materials whichmay serve as pharmaceutically acceptable carriers include: (1) sugars,such as lactose, glucose and sucrose; (2) starches, such as corn starchand potato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils such as castor oil,olive oil, peanut oil, macadamia nut oil, walnut oil, almond oil,pumpkinseed oil, cottonseed oil, sesame oil, corn oil, soybean oil,avocado oil, palm oil, coconut oil, sunflower oil, safflower oil,flaxseed oil, grapeseed oil, canola oil, low viscosity silicone oil,light mineral oil, or any combination thereof; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; (21) gums such as HP-guar; (22) polymers;and (23) other non-toxic compatible substances employed inpharmaceutical formulations.

The term “pharmaceutically acceptable salts” is art-recognized, andrefers to relatively non-toxic, inorganic and organic acid additionsalts of compositions of the present invention or any componentsthereof, including without limitation, therapeutic agents, excipients,other materials and the like. Examples of pharmaceutically acceptablesalts include those derived from mineral acids, such as hydrochloricacid and sulfuric acid, and those derived from organic acids, such asethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, andthe like. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, and nitric acids; and the salts prepared from organic acidssuch as acetic, fuoric, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, tolunesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic acids.

The term “topical” refers to a route of administration, i.e.,administering a drug to body surfaces such as the skin, tissues, ormucous membranes of a subject in need thereof. For example, topicalmedications may be administered to the eye lid, eye lashes, eye lidmargin, skin, or into the eye (e.g., ocular surface such as eye dropsapplied to the conjunctiva). Topical medications may also beinhalational, such as asthma medications, or medications applied to thesurface of a tooth.

The term “intraocular” as used herein refers to anywhere within theglobe of the eye.

The term “intravitreal” as used herein refers to inside the gel in theback of the eye. For example, a Lucentis injection is administeredintravitreally.

The term “subretinal” as used herein refers to the area between theretina and choroid. For example, iScience device is administeredsubretinally.

The term “intracapsular” as used herein refers to within the lenscapsule. For example, iVeena device is administered intracapsularly.

The term “suprachoroidal” as used herein refers to the area between thechoroid and sclera. For example, Clearside device is administeredsuprachoroidally.

The term “subtenon” as used herein refers to the area posterior to theorbital septum, outside the sclera, below tenon's capsule. For example,triamcinolone injections are administered to the subtenon.

The term “subconjunctival” as used herein refers to the area between theconjunctiva and sclera. For example, Macusight rapamycin injection isadministered to the subconjunctival area.

The term “intracameral” as used herein refers to “into a chamber” of theeye, for e.g., into the anterior or posterior chamber of the eye. Forexample, any injections during cataract surgery are administered tointracamerally.

The term “intrapalpebral” as used herein refers to into the eyelid. Forexample, Botox injections are administered intrapalpebrally.

The term “cul-de-sac” as used herein refers to the space between theeyelid and globe. For example, Ocusert device is administered to thecul-de-sac.

The term “retrobulbar” as used herein refers to behind the orbit of theeye. The term “peribulbar” as used herein refers to within the orbit oradjacent to the eye. For example, anesthetic block before eye surgery isadministered to the retrobulbar or peribulbar space.

As used herein, a “subject in need thereof” is a subject having adisorder which the hydrophobic drug described herein is intended to beused for treating, e.g., inflammatory disorders, respiratory disorders,autoimmune diseases or cancer A “subject” includes a mammal. The mammalcan be e.g., a human or appropriate non-human mammal, such as primate,mouse, rat, dog, cat, cow, horse, goat, camel, sheep or a pig. Thesubject can also be a bird or fowl. In one embodiment, the mammal is ahuman.

The term “preventing,” when used in relation to a condition, suchblepharitis, is art-recognized, and refers to administration of acomposition which reduces the frequency of, or delays the onset of,signs and/or symptoms of a medical condition in a subject relative to asubject which does not receive the composition.

The term “treating” is an art-recognized term which refers to curing aswell as ameliorating at least one symptom of any condition or disease.

EXAMPLES Example 1: Preparation of 0.1% Fluticasone PropionateNanoparticles

Methods: A HPLC Method to Determine the Concentration OffluticasonePropionate was Developed, with the Details Provided in A.

The specific composition of Phase I depends upon the solubility of thedrug in this phase. The solubility of fluticasone propionate inFDA-approved solvents and excipients was determined by dissolving 10 mgof drug in each solvent, vigorous vortexing and equilibrating overnightat 25 degrees centigrade. The suspension was centrifuged at 10,000 rpmand the supernatant analyzed by RP-HPLC at 239 nm. The solubility andcompatibility of fluticasone propionate in each of the solvents wasassessed.

A. HPLC Method Development

USP methods for the analysis of Fluticasone Propionate (cream, ointment)all utilize an extraction method with hexane, prior to dilution with themobile phase, most likely due to the presence of excipients that candegrade or block the column, lower resolution on peak separation andloss in peak height. Extraction methods result in loss of degradationproducts, especially those that have not been previously characterized.It was deemed necessary to develop a method that would result inquantitation of the API, as well as degradation products that may arisedue to potential incompatibilities with excipients.

Sample Preparation Method

1. A 400 μl sample (1 mg/ml drug suspension) was combined with 1.6 ml ofmobile phase and vortex mixed. (Sample now 0.2 mg/ml)

2. 2 ml of sample was retrieved in a 5 ml syringe then filtered by handpressure through a syringe MILLEX® GV filter (MILLIPORE®, 33 mmdiameter, 0.22 um, DURAPORE® (PVDF), cat #: SLGV033RB, yellow). Theeffort needs a moderate amount of hand pressure.

3. The filtered sample was injected directly on the HPLC using theisocratic method.

Column Washing:

After several injections of samples that contained the formulation thatwere processed using the new dilution/filtration method, the columnpressures did increase slightly from 222 bar to 230 bar. It was foundthat washing the column with mobile phase or a combination of methanoland 0.1M ammonium acetate solution at pH=7 was useful in reducing thecolumn pressures to original pressures of about 222 bar. With thecurrent column flow rate of 1.5 ml per minute and the long 250 mm columnpressures are expected to be higher than similar method with lower flowrates and shorter column lengths. The HPLC has a cut off pressure of 400bar. The monitoring the column pressures will be essential todetermining when column washing is required so the HPLC method nowrecords the pressures along with the scans. Also additional dilutioninjections, that do not contain the formulation, will be added morefrequently to wash the column and prevent over pressurization, poor peakshape and loss of height.

Sample Set-Up

A sequence to run multiple samples of the formulation should includeblank injections to prevent an increase in column pressure. When theaccuracy samples were run on the HPLC, 12 injections of vehicle weredone where the pressure increased from 221 bar to 230 bar. Theseinjections were then followed by 8 samples which did not contain anyvehicle and the pressure dropped to 228 bar. Additional washing was doneafter the sequence to drop the pressure to a lower level. Based on theseresults a total of 6 to 8 injections of the formulation prepared asdescribed should be followed by 2 to 4 injections of mobile phase.Additional column washing should be considered prior to anotherformulation sequence if needed.

Chromatography Conditions:

Instrument: AGILENT® 1200 HPLC with autosampler and DAD detector.

Mobile phase: Isocratic, 50% methanol, 35% 0.01M ammonium phosphatepH=3.5, 15% Acetonitrile.

Flow rate: 1.5 ml/min

Run time: 20 minutes

Column: PHENOMENEX LUNA® C18 5 micron 100A 250-4.6 mm P/N 00G-4041-EO

Column temperature: 40° C.

Sample tray: Room Temperature

Injection Volume: 50 micro liters

DAD detection: 239 nm

Sample setup: Blanks were run in the sequence between sets ofexperiments to ensure no carry over.

Standard preparation: A 5 mg/ml standard stock solution of fluticasonewas prepared by weighing up the solid and dissolving it in 100%acetonitrile. The dilution of this stock for the calibration curvesamples were done in sample diluent. (50% acetonitrile/water)

Sample diluent: 50% acetonitrile/water.

Method Development Aspects

Specificity

The peak shape and height and retention times of FP and its impuritiesshould be similar with the samples that contain vehicle or mobile phaseas the diluent. Table 1 below shows the comparison of peak areas andheights for HPLC samples that contain vehicle or only mobile phase,shown in FIG. 2 .

TABLE 1 FP Area and height Analysis. Vehicle Diluent (MP) Sample AreaHeight Area Height  0.153 mg/ml 10672.1 531.6 10639.7 561 0.2044 mg/ml14180.7 710.3 14288.15 753.7 0.2555 mg/ml 17864.6 894.45 17981.5 947.9

There is a very good match between the samples with and without theformulation vehicle. Table 2 shows the areas and heights of thesesamples.

TABLE 2 Heights and Areas with 50% ACN/Water Diluent 50%Acetonitrile/Water Sample Area Height 0.2112 mg/ml 11096.5 578.2 0.1976mg/ml 14781.2 767.6  0.264 mg/ml 18727.7 972.2

B, C and D Impurities:

The impurities B, C and D from the vehicle injections were also comparedwith the same impurities from the samples that did not contain thevehicle. Table 3 below shows equivalency between the two samples. Thediluent is mobile phase.

TABLE 3 Impurities B, C and D Vehicle Diluent (MP) Sample Impurity AreaHeight Area Height  0.153 mg/ml B 5.5 0.41 3.3 0.28 C 7.25 0.48 6.3 0.46D 7.35 0.5 7.2 0.49 0.2044 mg/ml B 4.2 0.4 4.4 0.37 C 9.3 0.52 8.3 0.6 D10.1 0.685 9.5 0.64 0.2555 mg/ml B 4.9 0.49 5.9 0.48 C 11.2 0.77 10.80.78 D 13.3 0.93 11.9 0.8

Retention Times

The retention times of fluticasone propionate and impurities B, C and Dare as follows:

TABLE 4 Retention Times of various sample preparations Vehicle MP 50%ACN/water Sample RT RRT RT RRT RT RRT FP 14.1 1 14.2 1 13.8 1 Imp B 7.80.55 7.8 0.55 7.5 0.54 Imp C 10.3 0.73 10.3 0.73 9.9 0.72 Imp D 11.70.83 11.7 0.82 11.6 0.84

Linearity

The linearity of the new sample preparation was evaluated by spikingsamples of the blank vehicle with a known amount of fluticasonepropionate, dissolved in acetonitrile. Spikes of 300, 400 and 500 μl ofa 5.11 mg/ml fluticasone propionate were dissolved into 2 grams ofvehicle and diluted to 10 mls with mobile phase (MP). The mobile phasewas: 50% methanol, 35% 0.01M ammonium phosphate at pH=3.5 and 15%acetonitrile. The results are shown below in Table 5. The units of thex-axis are mg/ml of FP. The method is considered linear if thecorrelation coefficient or R2 value is 0.999 or greater.

TABLE 5 Linearity of fluticasone propionate in formulation vehicle FileSample Area Height Concen Area Slope Intercept Feb16B02 1^(st) 0.15310671 530.1 0.153 10672.1 70168.8628 −96.3653395 inject Feb16B03 2^(nd)10673.2 533.1 0.2044 14180.7 inject Feb16B04 1^(st) 0.2044 14169.7 708.80.2555 17864.6 inject Feb16B05 2^(nd) 14191.7 712.4 inject Feb16B061^(st) 0.2555 17870.3 893.3 inject Feb16B07 2^(nd) 17858.9 895.6 inject

The same spikes were also done using 10000 mobile phase. The linearityof these samples are shown below in Table 6. The x-axis in this case ismg/ml of fluticasone propionate.

TABLE 6 Linearity using mobile phase as diluent File Sample Area HeightConcern Area Slope Intercept Feb16B16 1^(st) 0.153 10637.5 560.2 .015310639.65 71627 −330.332 inject Feb16B17 2^(nd) 10641.8 561.8 0.204414288.15 inject Feb16B18 1^(st) 0.2044 14290.7 754.5 0.2555 17981.5inject Feb16B19 2^(nd) 14285.6 752.8 inject Feb16B20 1^(st) 0.255517980.4 947.6 inject Feb16B21 2^(nd) 17982.6 948.2 inject

Chromatograms of the above samples from the same concentrations ofvehicle and diluent samples were overlaid and show identical peak shapesand heights for fluticasone propionate and for impurities B, C and D.

Precision

Precision was evaluated by injecting a 0.2 mg/ml sample 10 times thatwas prepared from a sample of the suspension. The results are providedbelow in Table 7.

TABLE 7 Precision File RT Area Height Feb15B01 14.626 14017.6 650.2Feb15B02 14.631 14004.5 654.5 Feb15C00 14 604 13975.8 655.5 Feb15C0114.588 13971.5 656.93 Feb15C02 14.59 13962.4 658.2 Feb15C03 14.579 13955658.4 Feb15C04 14.569 13941.7 660.3 Feb15C05 14 566 13931.7 662 Feb15C0614.568 13935.4 665.4 Feb15C07 14.559 13935.4 664.6 Average 14.6 13963.1658.6 Std Dev 0.0 29.7 4.7 RSD 0.2 0.2 0.7

The target relative standard deviation (RSD) for a precision evaluationis ≤1.0%. All values were well within this range.

Accuracy

The accuracy of the method at 3 levels with the new sample preparationwas evaluated by spiking a known amount of fluticasone propionate intoabout 2 grams of vehicle and comparing the calculated with the actualresults. Table 8 below shows the recoveries using the calibration curveshown in Table 5.

TABLE 8 Spiked Samples Sample Area Av Area Calculated Actual Agreement360 12618.4 12617.2 0.181 0.184 98.5 12616 420 14803.7 14803.6 0.2120.215 98.9 14803.5 480 17063 17059.8 0.244 0.245 99.7 17056.6

The acceptance criterion on this case is spike recovery of 99 to 101%.In this case there is good correlation between the actual and calculatedvalues.

LOD and LLOQ

From the blank of this method the noise is approximately 0.1 absorbanceunits which is the same for the LOD and LLOQ calculations in Part A ofthis report. The LLOQ and the LOD should be 10× and 3×this heightrespectively. Since the peak heights are very similar with and withoutthe vehicle present, the LOD and LLOQ were prepared to the sameconcentration ranges as Part A of this report however in this case thespike concentration were prepared in mobile phase, spiked into 2 gramsof vehicle and diluted to 10 mls with mobile phase to the LOD and LLOQconcentrations. The samples were injected 2× and the averages are shownbelow. A sample of 511 ng/ml gave a reproducible area/height of31.4/1.7. (LLOQ). For the LOD, a sample of 1 53.3 ng/ml gave anarea/height of 8.1/0.44. The heights of both the LLOQ and LOD areapproximately what was calculated based on the measured noise.

B. Solubility Determination of Fluticasone Propionate

The solubility of fluticasone propionate is given in Table 9. Thespecific composition of Phase I depends upon the solubility of the drugin this phase. The solubility of fluticasone propionate in FDA-approvedsolvents and excipients was determined by dissolving 10 mg of drug ineach solvent, vigorous vortexing and equilibrating overnight at 25degrees centigrade. The suspension was centrifuged at 10,000 rpm and thesupernatant analyzed by RP-HPLC at 239 nm. The solubility andcompatibility of fluticasone propionate in each of the solvents wasassessed.

TABLE 9 Solubility of Fluticasone Propionate Solubility Solvent (mg/ml)Ethanol 4.4462 PEG 400 4.3310 Glycerin 0.1441 Propylene glycol 0.7635PHOSAL ® 50 PG (phosphatidylcholine 0.4261 concentrate with at least 50%phosphatitylcholine and propylene glycol) PHOSAL ® 53 MCT(phosphatidycholine 0.4000 concentrate with at least 53%phosphatitylcholine and ethylemethylketone) PHOSAL ® 50 PG(phosphatidycholine 0.6601 concentrate with at least 50%phosphatitylcholine and propylene glycol) Polysorbate 60 4.9099Polysorbate 80 4.6556 Methylene Chloride 9.2472 Polysorbate 20 7.0573SPAN 80 ® (sorbitan monooleate) 0.0521 SPAN 20 ® (sorbitan monooleate)0.0469 PPG 2.2269 n-octanol 0.0873 Corn oil 0.0069 Castor oil 0.0180Mineral oil 0.0000 oleic acid 0.0136 PEG 200 4.2060 Phos buff pH = 70.0095 Acetone 62.976 Dextrose 5% 0.0053 water 0.00014

C. Nanocrystal Preparation by Anti-Solvent Crystallization DuringSonication (1 Step Process)

The process is as shown in FIG. 3 , without the purification step. Inthe case of Fluticasone Propionate, the drug was dissolved in thefollowing composition: Fluticasone Propionate (0.45%), TWEEN 80®(polysorbate 80) (7.44%), PEG 400 (23%), Polypropylene Glycol 400(69.11%). This composition was Phase I. The solubility of FluticasonePropionate was maximized in each of these solvents. Table 9 was utilizedto arrive at the composition of Phase I. The final composition (afterPhase I is added to Phase II) contained the drug at 0.1% w/w and theexcipients at concentrations approved for ophthalmic medications.

Phase I and Phase II were both sterile filtered through 0.22 micron PVDFfilters before mixing. In an experiment investigating the drug bindingkinetics of fluticasone propionate in Phase I to the filter, it wasfound that there was little or no binding of FP with the PVDF filter.

Sterile Phase I was added drop-wise into a sterile continuous phase(Phase II solution) while sonicating. 4.3 g of Phase I was addeddrop-wise to 15.76 g of Phase II. Sonication was performed with a SONICRUPTURE® 400 (OMNI INTERNATIONAL®, Inc.). The sonication conditions wereas follows: (a) Tip size (12.7 mm), temperature 2-4° C., power output 10W, duration: 1.5 minutes, batch size was 20 ml. This was accomplishedusing a 50 ml beaker. The rate at which phase I was added to phase IIgoverns the particle size of the crystals formed. For the 20 ml batch,the rate at which phase I is added to phase II was 2.15 ml/min.

The specific composition of phase II is extremely nuanced, since thecomponents of this phase act as the stabilizing phase for the dropletsas the nanocrystals are being formed. The effectiveness of thestabilizer is dependent upon the molecular weight and chemical structureof the stabilizing polymer, its adherence to the drug surface and itsability to lower the surface energy of the nanocrystals. Additionally,the concentration of the polymer in the continuous phase appears toaffect the particle size of the suspension. The function of thestabilizing phase is to also, prevent coalescence of the droplets priorto formation of the nanoparticles. For the preparation of 0.1%fluticasone propionate, the final composition of Phase II was 0.013%benzalkonium chloride, 0.25% methyl cellulose and 99.7% water. Forfluticasone propionate, the suspension obtained at the end of Step 1contains excipients at regulated amounts allowed in FDA approvedophthalmic medicaments. A 0.1% fluticasone propionate nanoparticlesuspension contains 0.1% drug, 3.23% TWEEN 80® (polysorbate 80), 4.97%PEG400, 14.95% PPG 400, 0.010% benzalkonium chloride, 0.38% methylcellulose and Q.S. purified water. The particle size range at this stepis 400-800 nm. The pH was 5.8 and the osmolality was 546 mOsm/Kg.

For the treatment of blepharitis, a hyperosmolal solution may betolerated, although an isotonic suspension is always desired, since theapplication is the interface of the eyelid and the ocular surface.

At a drug concentration of 0.06%, the vehicle composition is isotonic(316 mOsm/kg). At this drug concentration, the respective concentrationsof excipients in the continuous phase are 2.57% TWEEN 80® (polysorbate80), 2.99% PEG400, 8.97% PPG 400, 0.010% benzalkonium chloride andpurified water (Q.S.). The pH of this solution is 6.5. NaOH may be addedto adjust the pH to a neutral pH. This can be then diluted to lowerconcentrations of fluticasone propionate nanocrystals suspended in thevehicle. Table 10 shows formulations of fluticasone propionate preparedat concentrations 0.06%-0.001%.

TABLE 10 Concentrations 0-0.06% fluticasone propionate Concen- Concen-Particle tration tration Osmolality size (% FP) (mg/ml) FP Vehicle(mOsm/kg) pH (microns) 0.06 0.6 PEG400 316 7.01 1.09 0.01 0.1 (2.99%),310 7.02 1.08 0.001 0.01 PPG400 305 7.01 solution 0 0 (8.97%), 306 7.00solution TWEEN 80 ® (polysorbate 80) (2.57%), BAK (0.011%), MC (0.2%),water (QS), NaOH (pH adj.)

The solutions meet ophthalmic criteria of pH, excipient composition andosmolality. Formulations at concentrations greater than 0.06% haveosmolality values >350 mOsm/kg. One of the issues with this formulationis “Ostwald Ripening”, or growth of particle size. Growth in particlesize is observed, when there is dissolved fluticasone propionate. Theexcipients present in the formulation dissolve some of the drug in thecontinuous phase. This results in particle instability over long-termstorage.

a. Effect of Phase II Polymer Composition on Initial Particle Size

The composition of phase II is critical and not predictable to oneskilled in the art. The step of forming the particles is a collaborativephenomenon between dispersion and coalescence of the droplets prior toprecipitation. Further, the properties of the drug would need to bematched with the properties of the particle stabilizing polymer.

As shown in FIG. 5 , use of HPMC, PVA, PVP, pluronics, and mixturesthereof, produced particles that were greater than 1 micron in meandiameter. The combination of 2% TWEEN 20® (polysorbate 80) and 0.5% CMCin water as the phase II solvent appeared to produce particles that weresmaller (0.4-0.6 microns). These particles however, grew over time to asize of 1.2 microns. Use of high viscosity polymers such as xanthan gumat 0.5% produced particles that were very large (>20 microns).

Phase III (Combination of Phase I+Phase II): The combination of 0.12%benzalkonium chloride/0.25% methyl cellulose (15 cP)/water in Phase IIseemed to be the composition that produced the smallest particlesreproducibly (400-600 nm, 15 batches). The combination of phase I andphase II is phase III, in which the nanocrystals are formed, whilesonicating.

This phase III composition was also stable chemically for more than 4weeks at 40 degrees C. This combination of polymers also maintains theparticle size at its original size for 5-14 days.

b. Particle Size of Batches Obtained by Top-Down Techniques

A comparison was performed of particles produced by top-down techniquessuch as microfluidization, jet-milling, ultrasound sonication (wetmilling) and homogenization. As shown in FIG. 6 , the batches producedby these techniques produce particulates that were all greater than 2microns. Some of the particles were 8 microns in size. The particlesunder the microscope appeared broken and debris-like.

c. Effect of pH of Phase II on Initial Particle Size

PH appears to play a critical role in the initial particle size, asshown in FIG. 7 . When Phase II was pH balanced to pH 7-7.2 withphosphate buffer at 0.1% w/w, initial particle size was consistentlyhigher (1.0-1.3 microns). When the pH was left unbalanced, the particlesize was consistently between 500-800 nm. FIG. 7 shows the mean particlesize of batches produced that were pH balanced and ones that were not pHbalanced. The pH-unbalanced batches (n=3) were 5.5 for 0.1% Fluticasonepropionate and 6.5 for 0.06% Fluticasone propionate (n=3). This effectof pH on particle size was unanticipated and unpredictable to oneskilled in the art.

d. Effect of Molecular Weight of Steric Stabilizing Polymer in Phase IIon Particle Size

Molecular weight of the steric stabilizing polymer in phase II plays asignificant role in the particle size of the nanocrystals, as shown inFIG. 8 . For example, hydroxypropyl methylcellulose (HPMC) at 4000centipoises consistently produces particles that are larger than thoseproduced when HPMC at 45 centipoises are used.

e. Effect of pH on Particle Size Stability

The stability of the nanocrystals is controlled by the pH of phase III,which is formed by the combination of phase I and phase II. A 20 grambatch of nanocrystals were produced at pH 5.5 and placed on stability at25 degrees C. Another 20 gram batch was produced at pH 7.5 and stabilitydetermined at 25 degrees C. for 30 days. Unexpectedly, the particles at7.5 grew rapidly to an average particle size greater than 1 micron. SeeFIG. 9 . This phenomenon was verified for batches at the 50 gram scale.

f. Final Composition of Phase III Product (Phase I+Phase II)

The composition of phase III is 0.1% fluticasone propionate, 1.63% TWEEN80® (polysorbate 80), 5% PEG400, 15% PPG400, 0.01% benzalkoniumchloride, 0.2% methyl cellulose and 77.95% water. The pH of this phaseis 5.5.

g. Purification of Nanocrystals of Fluticasone Propionate

Nanocrystals of fluticasone propionate were purified by exchange of thecontinuous phase by either tangential flow filtration or hollow fibercartridge filtration. A high flow membrane is used for the filtration.Filters such as PVDF, PES are appropriate for this purpose, at pore size0.22 microns or less. A tangential flow apparatus from MILLIPORE®(PELLICON® XL 50 system) can be used for this purpose.

For a batch size of 250 g, the nanocrystal suspension (Phase III) waspoured into the 500 ml reservoir under a pump speed of 3, with thepressure never exceeding 30 psi. When the nanosuspension was washed downto 10 ml, the washing fluid was added. The washing fluid was 0.1% TWEEN80® (polysorbate 80), fed into the reservoir at 30° C. The washing fluidwas exchanged twice to ensure complete exchange of the buffer. Theconcentrate was then assayed for drug concentration. Based on the assayresults, the reconstitute volume was adjusted to achieve the desiredconcentration. Additionally, methyl cellulose, sodium chloride, andphosphate were added to arrive at an osmolal composition.

As shown in FIG. 10 , the purified fluticasone propionate nanocrystalsdid not display any agglomeration over time.

Example 2: Exemplary Nanocrystal Manufacturing Process

The process to manufacture purified, stable, sterile nanocrystals offluticasone propionate of size range 400-600 nm includes:

an in-situ crystallization step, whereupon a sterile phase I solution offluticasone propionate in PEG400, PPG400, and TWEEN 80® (polysorbate 80)is mixed under sonication, at a flow rate between 1-1.4 ml/min with asterile phase II solution comprising methyl cellulose between 15 cP-45cP, benzalkonium chloride and purified water in the ratio 0.2-1 and pHbetween 5-6, to produce a sterile phase III suspension; and

an annealing step, whereupon the fluticasone propionate nanocrystals inphase III are held in a holding tank in the temperature range of 25-40degrees centigrade for a duration range of 30 minutes to 24 hours; and

a purifying step, whereupon the fluticasone propionate nanocrystals arewashed by exchange filtration through a membrane of pore size 0.1-0.22microns by a sterile aqueous solution comprising of 0.1-0.5% TWEEN 80®(polysorbate 80); and

a concentration step, whereupon the fluticasone propionate nanocrystalsare concentrated to a range between 0.0001%-10%; and

a final formulation step, whereupon additional excipients are added insterile form to meet FDA and drug product criteria of osmolality, pH,viscosity, biocompatibility and permeability deemed appropriate for theparticular product and clinical indication.

Example 3: Nanocrystal Manufacturing Process-Batch Process

The process described in this Example was applied to produce FP crystalsin a size range of 400-600 nm. Particle size optimization using thisprocess is a function of phase I and II composition, sonication outputenergy, flow rate of phase I, temperature of phase I and II. The flowrate of phase I for all batches (20-2000 g) was 1.43 ml/min.

The composition of phase I: FP: 0.45% w/w; TWEEN 80® (polysorbate 80):7.67% w/w; PEG 400: 23.18% w/w, PPG400 (PPG=polypropylene glycol):68.70% w/w. The composition of phase II: benzalkonium chloride: 0.020%w/w, methyl cellulose 15 cp 0.40% w/w, water (QS to 100%). Thecomposition of phase III dispersion: FP: 0.225% w/w, TWEEN 80®(polysorbate 80): 3.796% w/w, PEG400:11.577% w/w, PPG400: 34.41% w/w,benzalkonium chloride 0.01%, methyl cellulose (MC 15 cP): 0.2% w/w,water Q.S. to 100%. The volume ratio of Phase I to Phase II was 1:1 forthis batch process.

The temperature of each phase I and II was 0-1° C. (ice water slurry).The sonication output energy was 25% using a ¾″ probe and an OMNI®CELLRUPTOR® Sonicator. The pH of phase II was 5.5. Higher pH resulted inlarger particles. It was also observed that at pHs <5, particle sizeswere between 150-220 nm, but the drug began to degrade at the lower pHs.

Similar to Example 1, it was found that the size of the FP crystals wascontrolled by selecting proper stabilizers and pH values of the phase IIsolution. See, e.g., FIGS. 7 and 8 .

A particle size range of 400-600 nm was achieved with lower temperatures(FIG. 11 ). Particles produced at room temperature were large andaggregated, indicating soft amorphous regions.

After fluticasone propionate crystals are prepared bysonocrystallization, the dispersion (phase III) was annealed at 25° C.The particles equilibrated to a steady particle size after at least 8hours of annealing time (FIGS. 12 and 13 ). This annealing stepunexpectedly, decreased the particle size. As shown in FIGS. 12 and 13 ,equilibrated particle size plateaus at 8 h and there is no statisticaldifference between different annealing temperatures, i.e., 4, 25 and 40°C. Further, the annealing effect is consistent for FP at concentrationsof 0.1% and 10%.

The crystals produced by the above process were purified, either bytangential flow filtration or by continuous centrifugation. A lab scalePELLICON® XL50 filtration apparatus was used to develop the filtrationconditions. The purpose of this step was to purify the crystals producedin the previous steps. FIGS. 14 and 15 showed that the drug loss usingPVDF filters with a 0.1 micron pore size was minimal. Purification bycentrifugation was accomplished by exchanging out the fluid with asolution of 0.1% w/w.

The final composition of fluticasone propionate was 0.0001-10% w/w,methyl cellulose 0.2% w/w (4000 cP), benzalkonium chloride 0.01% andwater (Q.S.). The final formulation is flexible in that additionalexcipients can be added to the formulation, depending upon theindication.

Example 4: Dispersability of Nanocrystal from Batch Process

It was observed that the final compositions or formulations of FPproduced in Example 3 remained dispersed over at least 8 hours. Inparticular, 5 ml of nanosuspension was placed in 10 ml glassscrew-capped vials, all of which contained 0.1% FP nanosuspension in thefinal composition. Each vial was shaken 10 times top over bottom todisperse the sample well. After shaking, each vial was stored at 25° C.and sampled over time to 24 hours.

Each sample was redispersed after 24 hours and re-sampled (shown by theblue arrows in FIGS. 16 and 17 ). Sampling was performed by taking a 0.5ml sample from the middle of the formulation. Samples were analyzed byassay by HPLC. As shown in FIGS. 16 and 17 , the final formulationsremain dispersed to at least 8 hours and re-disperse well on shaking.Also, concentrations 0.005%-10% FP all re-dispersed well, andre-dispersability was reproducible across the batch scales (20 g-2000g). All concentrations were more than 80% dispersed at 24 hours at RT.All concentrations re-dispersed with shaking of vial, indicating aflocculated robust suspension. It was concluded that higherconcentrations do not result in a faster rate of settling.

Example 5: Stability of Nanocrystal from Batch Process

It was also observed that the final compositions or formulations of FPwere stable across all concentrations tested, i.e., 0.005%, 0.01%, 0.1%,and 10%. Samples were placed in 4° C., 25° C., 40° C. stabilitychambers. Stability time-points: T=0 d, T=1 week, T=2 weeks, T=4 weeks.

Assay by HPLC showed that: 99-101% for 4° C., 25° C. and 106% for 40° C.There were no changes to impurities B, C and D in the samples testedfrom T=0 d. The pH (6.5-6.8) of the formulations tested did not changefrom T=0 d. Further, the FP particle size (505-620 nm) also did notchange from T=0 d.

Example 6: Uniformity of Nanocrystals Composition

A new suspension formulation for fluticasone propionate (FP) containingsodium chloride, phosphate, methyl cellulose, TWEEN 80® (polysorbate80), benzalkonium chloride and water was tested for content uniformityover time by sampling the top, middle and bottom of the suspensionsolution. The purpose was to determine the length of time the suspensionparticles remained equally distributed in solution after shaking.

About 20 ml of a 0.07% FP suspension was put into a vial and shaken 10times up and down to suspend the FP particles. 200 μl samples were takenof the top, middle and bottom at 0, 0.5, 1, 3, 6.5 and 23 hours. All ofthe samples were analyzed by HPLC using a calibration curve. The sampleswere taken directly into an HPLC vial and diluted with 800 μl of diluent(75/25 acetonitrile/water). The weights of the 200 μl sample and 800 μldiluent were recorded and used in the final calculation of the amount ofFP in each sample.

Results showed that there was little or no difference between the top,middle and bottom samples in the first 6.5 hours. The 23 hour samplehowever, visually had settled and was supported by the HPLC results.

Based on the dilution described above a three point calibration rangewas chosen from 0.056 to 0.45 mg/ml. See Table 11 below. Three standardsolutions of FP were prepared from a 0.5787 mg/ml stock standard.

TABLE 11 Preparation of Standard Solutions Wt of Total Concentration Wtof Vehicle (g) Weight of Weight of (mg/g) Stock (g) (200 ul) Diluent (g)sample (g) 0.05645 0.0822 0.1780 0.5825 0.8427 0.2813  0.4121 0.18910.2467 0.8479 0.4506  0.6579 0.1870 0    0.8449

A calibration curve was prepared using three known concentrations of astock solution as described above and 200 ul of the blank vehicle tocorrect for any matrix affects that the vehicle may have on thestandards.

Calculations for Concentrations were based on the formula:(Wt of stock)×(Stock Standard)/(Total wt of sample)

The calibration curve is shown in Table 12 below. All of the standardsare in mg of FP per grams of solution.

TABLE 12 Fluticasone Propionate Calibration Curve Data Standard AverageConcen- Area # tration Area (injection 1, injections (mg/g) Countsinjection 2) Slope Intercept 1 0.05645 3731.8 3729.45 65428.9275837.85164626 2 3727.1 1 0.2813  18448 18447.35 2 18446.7 1 0.4506 29517.1 29517.65 2 29518.2 Datafit: R² = 1

Using the calibration curve in Table 12, the time point samples wereanalyzed using the slope and intercept. Table 13 below shows the dataobtained from the time-point sample analysis.

TABLE 13 Time Point analysis Con Wt of Wt of (mg/g) Wt of FP in 200 ulof HPLC HPLC of Con of Sample Area HPLC Sample Sample layer layer(Hours) (HPLC) Sample (g) (mg) (g) (mg/g)   0 h-Top 9310.8 0.1417 0.83930.119 0.1779 0.6686   0 h-Middle 9842.3 0.1498 0.8574 0.128 0.19270.6667   0 h-Bottom 10312.2 0.1570 0.8649 0.136 0.2007 0.6767 0.5 h-Top9233.2 0.1405 0.8397 0.118 0.1764 0.6690 0.5 h-Middle 10364.8 0.15780.8659 0.137 0.2054 0.6654 0.5 h-Bottom 10324.1 0.1572 0.8653 0.1360.2015 0.6751   1 h-Top 9142.1 0.1391 0.8329 0.116 0.1736 0.6676   1h-Middle 10089.1 0.1536 0.8611 0.132 0.2002 0.6608   1 h-Bottom 10883.20.1658 0.877 0.145 0.2163 0.6721   3 h-Top 9268.7 0.1411 0.8397 0.1180.1787 0.6629   3 h-Middle 9454.8 0.1439 0.8471 0.122 0.1874 0.6506   3h-Bottom 10351.5 0.1576 0.875 0.138 0.2136 0.6457 6.5 h-Top 9588.20.1460 0.8504 0.124 0.1879 0.6606 6.5 h-Middle 9555.9 0.1455 0.85530.124 0.1935 0.6430  65 h-Bottom 10128.3 0.1542 0.8665 0.134 0.20510.6515  23 h-Top 2479.1 0.0373 0.8478 0.032 0.1868 0.1693  23 h-Middle4041.1 0.0612 0.8507 0.052 0.1859 0.2800  23 h-Bottom 27409.7 0.41830.867 0.363 0.2034 1.7832

The data was also graphed over the entire time point range and was shownin FIG. 18 .

Example 7: Nanocrystal Manufacturing Process-Flow Process

Nanosuspensions of fluticasone propionate at a particle size range of400-600 nm were also prepared using a flow process scheme.

Fluticasone propionate nanosuspensions were prepared using the flowreactor shown in FIG. 19 . As shown in the flow schematic in FIG. 4 ,phase I and phase II were metered into the flow reactor.

The particle sizes of these nanosuspensions were measured with MALVERN®ZETASIZER® 590. Both Phase I and Phase II solutions, which were used formaking nanosuspensions, were pumped continuously into the sonicator flowsystem. 25 batches of samples were prepared under a variety ofconditions. The impact of the flow rates of both phases, the annealingtemperature of Phase III, and the amplitude of sonicator on particlesizes, was analyzed. Most aspects of “batch process variables” asdescribed in Examples 1 and 3 still applied, such as the temperature ofmixing two phases, type and viscosity/molecular weight of the cellulosicstabilizer in phase II, pH of phase II, and the annealing temperatureand time.

Materials and Equipment:

(A) Raw ingredients were listed in Table 14 below

(B) MALVERN® NANOSIZER® S90

(C) Flow Reactor

(D) Sonicator probe, size 25 mm. 1″ with probe extender

(E) Pump I (NE-9000, New Era Pump Systems Inc.)

(F) Pump II (Console Drive, Cole-Palmer)

TABLE 14 Excipients/drug Manufacturer Fluticasone HOVIONE ® Methylcellulose (15 cP) SHINETSU ® Benzalkonium chloride (BKC) SIGMA-ALDRICH ®Polypropylene glycol 400 ALFA AESAR ® Polyethylene glycol 400 SPECTRUMCHEMICAL ® TWEEN 80 ® (polysorbate 80) SPECTRUM CHEMICAL ®

Both Phase I and Phase II solutions were prepared in advance before theywere pumped into the flow system at 1:1 ratio. The preparation detailsand the compositions of both phases are described below, with 500 gbatch as an example.

Preparation of Phase I (500 g Batch)

2.28 g of Fluticasone propionate was gradually added into a solution of38.34 g of TWEEN 80® (polysorbate 80), 116 g of PEG 400, and 344 g ofPPG 400. The solution of all components was vortexed and ultrasonicatedusing a standard sonication water bath until all of the solids went intosolution.

(grams) (%) Fluticasone Propionate 2.282 0.46 TWEEN 80 ® (polysorbate80) 38.337 7.66 PEG 400 115.994 23.17 PPG 400 343.987 68.71

Preparation of Phase II (500 g Batch)

1 g of 10% benzalkonium chloride solution was added into 299 g of waterand 200 g of 1% methyl cellulose (15 cP) mixture. The mixture wasvortexed. The composition of Phase II was as follows: benzalkoniumchloride 0.020%, methyl cellulose 15 cp 0.4%, water 99.58%.

Mixing Conditions of Phase I and Phase II (500 g for Each Phase: Totalof 1000 g of Phase III)

The conditions for the mixing step are listed below:

Temperature of the mixture of Phase I and Phase II: 0˜5° C.

Ultrasonicator tip size: 25 mm in diameter

Ultrasonicator amplitude: 25˜75% (depending on the specific experiment)

Flow rate of Phase I: 12˜700 ml/min (depending upon the specificexperiment)

Flow rate of Phase II: 12˜700 ml/min.

Chiller temperature: 0˜−10° C.

Cooling air: 5 psi

Experiment duration time: 2˜8 min.

Mixing Procedures (500 g Batch for Each Phase)

250 g Phase II was loaded into the sonicator. Chiller (0˜−10° C.) andcooling air (5 psi) were then turned on. 500 g of Phase I was added intoa 1000 ml beaker that sat in an ice/water mixture bath. The remaining250 g of Phase II was added into another 1000 ml beaker that sat in anice/water mixture bath. The temperature of each phase was stabilized forat least 30 minutes. The pump flow rates of each of the two phases wereset as 12˜700 ml/min. Then the ultrasonicator was turned on andamplitude adjusted. Turned on the pumps. Once both phases were pumpedin, stopped the ultrasonication, pumps, and air generator.

25 batches of samples were prepared under a variety of conditions. Mostbatches have peak mean particle sizes below 1 micron, except threebatches that were prepared at relatively high flow rates (e.g., 700ml/min for each phase and 250 ml/min for each phase).

The Impact of Flow Rates of Both Phases on Particle Sizes

Both phases were pumped at same actual flow rate (ratio of Phase: II was1). The particle sizes (represented by square dots in FIG. 20 ) wereplotted against the final flow rates (represented by vertical bars inFIG. 20 ) of Phase III in FIG. 20 . Three samples prepared with 200ml/min have the smallest particle sizes about 400-600 nm.

These experiments demonstrated that fluticasone propionate nanocrystalscould be prepared using the flow process schematic shown in FIG. 4 .Microscopic examination demonstrated plate-like morphology for thecrystals. Preliminary stability studies on formulations prepared usingthe flow process (4 week stability at 25 and 40 C) showed stability ofparticle size and chemical integrity.

In general, trends were noted, as to the process variables that controlparticle size. Control of temperature of phase I and phase II to <2° C.led to consistent and robust production of uniformly sized particles.Other variables were the output energy of the sonication and flow ratesof phase I and phase II. Flow rates appeared to be the controllingvariable in generating particle sizes of uniform range. With the currentsonicator probe design, the highest flow rates that achieved theparticle size range of 400-600 nm was ˜200 ml/min/pump, or 400 ml/minfor phase III.

Example 8: Additional Characterization of Nanocrystals Manufactured byBatch Process

Nanocrystals of FP were prepared using a 1000 g batch process similar tothat described in Example 1 or 3. The suspensions were collected intosolids by centrifugation and dried in a vacuum oven for 12 hours. Twoadditional batches (i.e., b and c) were prepared using the same process.

Homogenized FP particles were prepared using a POLYTRON® (KINEMATICAAG), speed setting 4 in an aqueous dispersion. The samples were washedusing a centrifugation process and dried in a vacuum oven.

Fluticasone propionate stock was used as received from the manufacturer.

Particle Size Assessment

Particle size of FP nanocrystals prepared by the batch process wasmeasured by a MALVERN® ZETASIZER® S90. The particle sizes of batches (b)and (c) were measured by a MALVERN® MASTERSIZER S®. As shown in FIG. 21, the nanocrystals produced by the batch process produced a narrowdistribution of crystals, within the size range 400-600 nm, whereas thestock FP material and the homogenized FP material had a broad particlesize distribution (FIGS. 21B and 21C respectively).

Fluticasone Propionate Crystal Suspension is Highly Stable

Nanocrystals prepared by the batch process were tested on stability, toassess if the particle size distribution remained with a narrow range of400-600 nm. The nanoparticles were formulated into a final vehicle thatwas comprised of 0.1% w/v FP, 0.90% w/v Sodium Chloride, 0.51% w/vMethyl Cellulose (MC 4000 cP), 0.10% w/v Sodium Phosphate, 0.20% w/vTWEEN 80® (polysorbate 80), 0.01% w/v Benzalkonium Chloride and 98.18%w/v water. The formulations were placed in stability incubators at 25°C. and 40° C.

Samples were measured for particle size, pH, osmolality and assay. Allsamples maintained pH, osmolality, particle size and assay [FP] over 75days at 25° C. and 40° C. FIG. 22 shows stability of particle size over75 days, even at 40° C.

This data suggest that fluticasone propionate prepared by the process ofthe invention is comprised of highly crystalline crystals and is of astable morphological microstructure, evidenced by the absence of crystalgrowth over time (Ostwald Ripening).

Saturated Solubility and Rate of Dissolution

The saturated solubility of FP was measured by IPLC for the nanocrystalsproduced by the batch process of the invention, FP homogenized and FPstock material. The saturated solubility for all three materials was40-45 μg/ml. In another study, the rate of dissolution of thenanocrystals (size range 400-600 nm) was compared to a batch thatcontained suspended and micronized fluticasone propionate in the sizerange 1-5 microns. The comparative rates of dissolution are shown inFIG. 23 .

The purity of the fluticasone propionate nanocrystals was assessed andcompared to the purity of the FP stock material as received from themanufacturer. Shown in FIG. 24A is the chromatogram of fluticasonepropionate drug substance (retention time: 13.388 minutes) and its knownimpurities (shown at retention times 6.457 minutes and 9.720 minutes).Shown in FIG. 24B is the chromatogram of fluticasone propionatenanocrystals produced by the batch process. In comparison with the stockdrug substance, fluticasone propionate nanocrystals produced by thebatch process was of higher purity, with marked absence of theimpurities at 6.457 and 9.720 minutes. Note that the scale for the HPLCchromatogram for fluticasone propionate crystals produced by the batchprocess was 0-500 mAU, compared to 0-1200 mAU for the stock material.Accordingly, it is concluded that the process of nanocrystallization andpurification of the invention creates purer nanocrystals of fluticasonepropionate.

Morphology of FP Nanocrystals

Shown in FIGS. 25A and B are optical micrographs (Model: OMAX, 1600×) ofdried fluticasone propionate crystals prepared by the batch process andcompared to FP, stock material. The appearance of the FP crystalsproduced by the nanocrystallization process is markedly differentiatedfrom the fluticasone propionate drug substance, stock material. As seenin FIG. 25A, fluticasone propionate nanocrystals are rod-shaped, with adefined oriented geometry. In contrast, the stock material offluticasone propionate did not appear to favor any specific shape orgeometry.

The external appearance and morphology of FP crystals prepared by thebatch process were compared to FP, stock material. Scanning ElectronMicrographs were collected at 10,000× magnification using a HITACHI® SEMinstrument. The experiments were performed at Microvision, Inc.,Chelmsford, Mass.

Visually, the differences between the crystals produced by the batchprocess and the other samples are striking. The fluticasone propionatecrystals prepared by the batch process were blade-like plates, or rodswith a defined oriented geometry (FIGS. 26A and 26B). In contrast, themorphology of fluticasone propionate stock crystals appeared rounded,not plate-like or with angled edges as the fluticasone propionatecrystals produced by the batch process (FIG. 27A).

FIG. 27B is the scanning electron micrograph of the homogenizedparticles of FP (top-down process). Visually, these particles appearedsimilar to the stock material.

Thermal Characteristics

To measure the thermal properties for each fluticasone propionatespecimen, approximately 10 mg was collected from each specimen andplaced in a clean alumina crucible. The table below summarizes thetesting conditions and parameters for the simultaneous thermal analysistests. The samples were (a) Fluticasone Propionate nanocrystals, and (b)Fluticasone Propionate, stock material. The specimens were tested undera heating rate of 10° C./min starting at 30° C. until reaching a finaltemperature of 350° C. This process was repeated for each specimen. Theexperiments were performed at EBATCO, LLC, Eden Prairie, Minn.

TABLE 15 Simultaneous Thermal Analysis Testing Conditions and ParametersSamples Fluticasone Propionate Stock, Fluticasone Propionate Crystalsproduced by the batch process Test instrument STA 449 F3-JUPITER ®Crucibles Alumina (Al₂O₃) Heating Rate 10° C./min Initial Temperature30° C. Final Temperature 350° C. Purge Gas Nitrogen, 20 mL/minProtective Gas Nitrogen, 30 mL/min

Thermal analysis test results are shown for each sample, in Table 16below. The softening temperature of a substance, also known as the glasstransition temperature was significantly lower for the fluticasonepropionate stock material (57.6° C.) compared to the fluticasonepropionate crystals produced by the batch process. Additionally, theheat of melting for the fluticasone propionate crystals produced by thenew process was significantly higher (54.21 J/g) than the FP stockmaterial (48.44 J/g), indicating that the former was a more crystallinematerial, requiring more energy to break inter-molecular bonds such asionic and hydrogen bonds.

TABLE 16 Glass Melting Latent Mass Transition Temperature Heat of ChangeUpper Limit Range Melting Specimen (%) (° C.) (° C.) (J/g) FPnanocrystals −46.12 63.5 10.1 54.21 FP Stock Sample −47.96 57.6 11.048.44

FIG. 28A shows the combined DSC/TGA of fluticasone propionate crystalsproduced by the batch process. In comparison with the thermalcharacteristics of fluticasone propionate stock material (FIG. 28B), theonset of melting of the FP nanocrystals was higher than the onset ofmelting of the fluticasone propionate stock: onset_(melting) (FPnanocrystals from batch process) 299.5° C.> onset_(melting) (FP, stock)297.3° C. Additionally, as evidenced by thermo-gravimetric (TGA), theonset temperature_(mass loss) (FP nanocrystals from batch process) 299°C. is higher than the onset temperature_(mass loss) (FP, as is) 250° C.The data suggest that the fluticasone propionate crystals produced bythe batch process have thermal behavior indicative of material morecrystalline and ordered than the fluticasone propionate stock material.

Fluticasone Propionate Crystals Prepared by the Batch Process are notSolvates or Hydrates

Theoretically, when solvents are entrapped in the crystal structure,they are termed “solvates”. When the specific solvent is water, thecrystals are termed “hydrates”. Solvates and hydrates of a particularcrystalline form display different properties such as dissolution,density, etc. Differential Scanning Calorimetry (DSC) can be used todetect the presence of an entrapped solvent, which can be induced toescape from the crystal lattice when heated. For crystals preparedutilizing the batch process, there were no additional melt transitions(DSC) or multi-phasic mass loss (TGA) (FIG. 28A) denoting that thecrystals were pure crystals, not solvates or hydrates. FluticasonePropionate stock material was also not a solvate or a hydrate, but ofcrystalline structure, as expected (FIG. 28B).

Fluticasone Propionate Crystals Produced by Batch Process have HigherBulk Tap Density Compared to Fluticasone Propionate Stock Material

The tap density of dried fluticasone propionate crystals prepared by thebatch process was 0.5786 g/cm³. In contrast, the tap density offluticasone propionate stock was 0.3278 g/cm³. The data suggest thatfluticasone propionate crystals produced by the batch process have ahigher packing than the stock fluticasone propionate.

Fluticasone Propionate Crystals Produced by Batch Process are notAmorphous or Partially Amorphous

It is to be noted that the fluticasone propionate crystals produced bythe batch process do not display “cold crystallization”, orcrystallization or amorphous phases prior to melting. Presence of asingle, sharp melt transition at 299.5° C. suggests lack of an amorphousor amorphic phase in the material. The sharpness of the melt transition(melting range 10° C.) also denotes a highly ordered microstructure. Incontrast, fluticasone propionate stock material melted over a slightwider range (11.1° C.).

Fluticasone Propionate crystals produced by the batch process andfluticasone propionate stock material were compared with each other withrespect to their infrared vibrational frequencies (FTIR), using aNICOLET® Fourier Transform Infrared Spectrophotometer. FTIR is utilizedto confirm/verify identity of a known organic substance, since specificbonds and functional groups vibrate at known frequencies. The FTIRspectrum of fluticasone propionate crystals produced by the batchprocess did not show presence of any additional vibrational frequencies(FIG. 29 ), when compared to the known FTIR spectrum of fluticasonepropionate (FIG. 30 ).

Crystal Structure of Fluticasone Propionate Produced by the Process ofthe Invention Vs. The Two Known Forms of Fluticasone Propionate

Polymorph 1 and polymorph 2 are the two crystal forms of fluticasonepropionate published previously. See, e.g., U.S. Pat. No. 6,406,718 B1and J. Cejka, B. Kratochvil and A. Jegorov. 2005. “Crystal Structure ofFluticasone Propionate”, Z. Kristallogr. NCS 220 (2005) 143-144. Frompublished literature, polymorph 1 is the most stable known form offluticasone propionate, in that it is the most abundant. Polymorph 1 isformed by free crystallization from solvents of medium polarity(acetone, ethyl acetate and dichlorimethane). Polymorph 2 crystallizesfrom supercritical fluid and only described in U.S. Pat. No. 6,406,718B1, with no other published accounts.

The crystal structure of polymorph 1 is provided in Cejka, et. al, withthe following unit cell characteristics: C₂₅H₃₁F₃O₅S, monoclinic, P12₁1(no. 4), a=7.6496 Å, b=14.138 Å, c=10.9833 Å.

The crystal structure of polymorph 2 is provided in U.S. Pat. No.6,406,718 B1 and Kariuki et al, 1999. Chem. Commun., 1677-1678. The unitcell lattice parameters are a=23.2434 Å, b=13.9783 A and c=7.65 Å. Theunit cell was described as orthorhombic. As noted in Kariuki et. al,there were striking similarities between the two crystal structures. Forreference, the calculated XRPD powder patterns of polymorph 1 (red) andpolymorph 2 (blue) are shown in FIG. 31B.

In the first set of studies to determine the crystal structure offluticasone propionate nanocrystals prepared by the batch process tocompare with the crystal structure of fluticasone propionate stockmaterial, X-Ray Powder Diffraction (XRPD) patterns of both materialswere collected by X-Ray Diffractometer (Shimadzu XRD 6000 Diffractometeroperating at 40 KV and 30 mA. The samples were split and pulverized foranalysis. The samples were scanned from 10 to 65 degrees two-theta 0.02°steps at 2 seconds per step. Diffracted x-rays were collimated using a0.05° receiving slit and detected with a solid state scintillationdetector. Peak intensity and resolution calibration were verified usingsolid quartz standard 640 d. These studies were performed at XRDLaboratories, IL.

The XRPD patterns of both Fluticasone Propionate crystals prepared bythe batch process and Fluticasone Propionate stock material werecompared with calculated XRPD patterns from the published crystalstructures of Polymorph 1 and 2. An overlay of the XRPD patterns ofFluticasone Propionate stock and Fluticasone propionate Polymorph 1indicated that the FP stock material existed as the polymorph 1, themost abundant and stable polymorph.

An overlay of XRPD patterns of FP crystals by homogenization (example ofa “top-down” process) and the FP stock material demonstrated excellent“peak-to-peak” agreement between the patterns, even the intensities. Itcan be concluded that the Fluticasone Propionate homogenized sample isof an identical polymorph as Fluticasone Propionate Stock (polymorph 1).In contrast, the XRPD pattern of fluticasone propionate crystals (batchprocess) was overlaid (black) on that for published polymorph 1 (red)and polymorph 2 (blue), there were clear differences in the diffractionpattern, shown in FIG. 31B. Further experiments performed at TriclinicLabs, Inc. determined the unit cell structure of the crystals producedby the batch process and the microstructural differences with standardpolymorph 1. The data suggest that fluticasone propionate crystalsproduced by the new process had a novel and differentiatedmicrostructure than standard polymorph 1.

Unit Cell Structure of Fluticasone Propionate Nanocrystals Prepared bythe Batch Process

All samples were prepared by filling the sample holder cavity withpowder and gently pressing the sample to give a flat reference surface.Any excess material was removed and returned to the original container.All measured data sets were pre-processed to remove background andscaled to a common area of 100000 counts over the common measurementrange. Indexing is the determination of crystal unit cells usingmeasured diffraction peak positions. Peak positions for the providedXRPD data files were initially determined using WINPLOT R®.

To model the peak intensity differences between the XRPD data sets (FP,batch process and Polymorph 1), a crystalline harmonic preferredorientation function was added to the crystal structure description, totest the hypothesis that the FP (batch process) were a novel crystallinehabit. The allowed harmonic symmetries were 2/m and ‘fiber’ using 8harmonic terms in the expansion. With the preferred orientation functionadded to the crystal structure description of standard polymorph 1, theXRPD patterns of standard polymorph 1 and fluticasone propionatecrystals produced by the batch process could be matched. This provedthat FP (batch process) was a novel crystalline habit of polymorph 1.

By definition, a crystalline habit of a known polymorph has differentmicrostructure such as planes of orientation, etc. (Miller Indices) thatcan lead to a different shape and appearance, while having the same unitcell structure and type. In the case of fluticasone propionate producedby the batch process, the crystals had a different appearance(demonstrated by SEM in FIG. 26 ) than the stock material (FIG. 27 ).

The differences between the XRPD data collected on micronized andproprietary batches of FP crystals were essentially differences indiffraction peak intensity. Peaks with non-zero ‘1’ Miller indices wereseen to significantly increase in intensity for the proprietarymaterial. Rietveld modeling of the proprietary material confirmed thatwithin the reflection powder samples, the FP nanocrystals from the batchprocess were strongly aligned with the [001] (c-axis) crystallographicdirection normal to the sample surface. This suggests that awell-defined crystalline habit is produced by the proprietary productionmethod and that the habit is most likely plate or blade like in nature.The proprietary material packed differently in the XRPD sample holder,due to the consistent habit, leading to the observed preferredorientation (PO). On the other hand, the stock material did not exhibitany significant preferred orientation (PO).

The effective crystal structure derived for the proprietary materialfurther suggests a blade or plate like habit with the crystallographica-b plane lying almost parallel to the largest exposed surface. Theeffective crystal structure can be used to investigate the functionalgroups of the API exposed by the largest crystal face of the bladehabit.

The unit cell structure of the fluticasone propionate crystals producedby the batch process is Monoclinic, P21, a=7.7116 Å, b=14.170 Å,c=11.306 Å, beta=98.285, volume 1222.6. In comparison, the crystalstructure of polymorph 1 is provided in Cejka, et. al, with thefollowing unit cell characteristics: C₂₅H₃₁F₃O₅S, monoclinic, P12₁1 (no.4), a=7.6496 Å, b=14.138 Å, c=10.9833 Å.

Thus, it can be stated that the fluticasone propionate (via batchprocess) is a novel crystalline habit which occupies a similar unit celltype as polymorph 1, which is the most stable and most abundant crystalstate published to date. Since the most stable polymorphs havetheoretically the highest melting point, it can be deduced that thenovel crystalline habit (fluticasone propionate via the process of theinvention) may be the most stable crystal structure of the drugsubstance discovered to date. As mentioned above, the melting point ofthe novel crystals was 299.5° C., as opposed to 297.3° C. for the stockmaterial (polymorph 1), as shown in FIGS. 28A and 28B. Also, theexistence of the novel crystalline habit in FP nanocrystals produced bythe process of the invention was reproducible.

MAUD is able to produce ‘pole-figures’ for specific crystallographicdirections based upon the preferred orientation parameters derivedduring the Rietveld modeling. For each crystallographic axis selected,the pole figure illustrates the angular distribution of that crystalaxis about the surface of the reflection sample holder. For an idealpowder, all crystallographic axes will be randomly oriented giving apole figure with a uniform color. For a single crystal sample, eachcrystallographic axis will be oriented in a single direction. If thatdirection is normal to the sample surface then the pole figure will showa single high intensity spot in the center of the plot. The pole figuresderived from the XRPD data collected on the FP nanocrystals via batchprocess showed a single high intensity central pole for the [001]crystallographic axis. This is indicative of strong preferredorientation with the crystallographic c-axis being normal to the surfaceof the powder sample. One possible driving force for this strongpreferred orientation occurs if the crystalline habit is plate like orblade like. When packed into a reflection holder and pressed flat, theflat surfaces of the crystal tend to align parallel with the samplesurface (like sheets of paper). This suggests that for the FPnanocrystals from the batch process, the crystallographic c-axis isclose to normal through the largest flat crystal face. In contrast, polefigures calculated for the FP stock material showed a generaldistribution of crystallographic orientations more typical of a close torandomly oriented sample.

Example 9: Triamcinolone Acetonide (TA) Crystal ManufacturingProcess-Batch Process

Triamcinolone acetonide is a synthetic corticosteroid used to treatvarious skin conditions, relieve the discomfort of mouth sores and innasal spray form as an over-the-counter relief for allergic andperennial allergic rhinitis. It is a more potent derivative oftriamcinolone, being about 8 times as potent as prednisone. Its IUPACname is(4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS)-4b-fluoro-6b-glycoloyl-5-hydroxy-4a,6a,8,8-tetramethyl-4a,4b,5,6,6a,6b,9a,10,10a,10b,11,12-dodecahydro-2H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-2-one,with molecular formula of C₂₄H₃₁FO₆ and molecular mass of 434.5 g mol⁻¹.

Triamcinolone Acetonide Solubility

Triamcinolone Acetonide (TA), stock was used as received from themanufacturer. Solubility of Triamcinolone Acetonide (TA) was measured inpropylene glycol, polypropylene glycol, TWEEN 20® (polysorbate 20),TWEEN 80® (polysorbate 80), PEG 400.

Initially, 5 mg of the TA was added to 10 g of solvent; the mixture wasvortexed for 5 min, and sonicated for 10 min in a water bath. 1-5 mg ofTA was added when the initial amount dissolved in the solventcompletely—clear solution of TA in solvent. The process was continueduntil saturation solubility was reached. The solvent that provided thehighest solubility was chosen for further development as Phase I.

The solubility of TA was evaluated in various pure non-aqueous systemsin order to prepare Phase I. TA is practically insoluble in water. Thesolubility of TA in propylene glycol, polypropylene glycol, PEG 400,TWEEN 20® (polysorbate 20) and TWEEN 80® (polysorbate 80) was evaluated.Initially, 5 mg of TA was added to these solvents and the suspension wasvortexed and sonicated for 15 min in a water bath at 37° C. When the API(i.e., TA) dissolved, 1 mg of drug was added to the vial. This processwas continued until a preliminary estimation of the drug in all solventswas achieved. The solubility of TA in Propylene glycol, polypropyleneglycol, PEG 400, TWEEN 20® (polysorbate 20) and TWEEN 80® (polysorbate80) was 14, 8, 7, 5.5 and 4 mg/mL, respectively.

Preparation of TA Nanocrystals

Phase I

This is the phase that the drug is solubilized in. Phase I was preparedwith the highest concentration of API in a chosen solvent. Sincepropylene glycol exhibited as a better solvent, it was chosen forfurther development. The final composition of phase I: TA: 1.4% w/w, PG(PG=Propylene glycol). The batch size was 50 grams.

Phase II

The composition of phase II: Benzalkonium chloride: 0.0125% w/w, methylcellulose 15 cp 0.257% w/w, water (QS to 100%). Since the TA degrades athigher pH (see, e.g., Ungphaiboon S et al. Am J Health Syst Pharm. 2005Mar. 1; 62(5):485-91), 0.1% citric acid was added to lower the pH of thesolvent. The final pH Phase II was 3.91. The batch size was 100 grams.Phase II was cooled down to 0° C. in ice-water slurry.

Generation of Phase II and Annealing

This procedure generates 150 grams of Phase III. The combination ofphase I and phase II produces nanocrystals of API dispersed in avehicle. This dispersion is Phase III.

Phase III was prepared by metering 50 g of Phase I into 100 g Phase II.

50 grams of Phase I was filled into a 60 ml syringe fitted with a needlethat was 6 inches long and 18 gauge. 100 g of Phase II was poured into a250 ml beaker and cooled to 0° C., using an ice-water slurry. Sonicationwas performed using SONIC RUPTOR™ ultrasonic homogenizer (OMNIINTERNATIONAL®) at a setting of 20% intensity, using a titanium probethat was ¾ inches in diameter. The flow rate of Phase I was kept at 1.43ml/min. Phase III was collected in a 250 ml pyrex beaker. The resultantPhase III was a milky-white dispersion. The dispersion was annealed at25° C. for 4 hours in the 250 ml beaker, covered with parafilm. Thecomposition of phase III dispersion: TA: 0.41% w/w, PG: 32.86% w/w,benzalkonium chloride 0.01%, methyl cellulose (MC 15 cP): 0.2% w/w,water 66.93% w/w.

Purification

This slurry was subsequently subjected to centrifugation (3×) at 10,000rpm and 4° C. The following steps were performed:

The slurry was divided into 6 50 ml polypropylene centrifuge tubes at 25ml each. To each tube was added 25 ml of the “wash” solution. The washsolution consisted of 0.01 w/w % benzalkonium chloride and 0.2% w/wTWEEN 80® (polysorbate 80) in distilled water. Thus, the dilution was1:1.

The diluted slurry was centrifuged at 10,000 rpm and 4° C. for 90minutes, using a THERMO-SCIENTIFIC® IEC CL31R MULTI-SPEED®.

After pelletizing, the pellets were re-dispersed with the wash solution,filled to the 50 ml mark. The dispersion was centrifuged as describedpreviously.

After two washes, the pellets were consolidated into two 1.5 mlcentrifuge tubes and re-dispersed with ˜1 ml of the washing solution.The dispersion was centrifuged again using an EPPENDORF® Centrifuge5415D at 12,000 RPM for 12 minutes.

The pellets were collected and consolidated into a 50 ml centrifuge tubeand re-dispersed it in 40 ml of washing solution. Dispersion wasachieved by vortexing and then sonicating it in water bath for 15minutes at room temperature. The dispersion was centrifuged at 10,000RPM for 10 minutes.

The supernatant was decanted and the pellet was dried for 72 hours at RTusing vacuum oven (VWR International, Oregon, USA).

Example 10: Characterization of Ta Crystals Manufactured by Process-FlowProcess

Particle sizing was performed on the Phase III dispersion made inExample 9 above, after annealing. MALVERN® dynamic light scatteringequipment (Model S90) was used to determine the nanocrystal size andsize distribution. To measure the particle size, 40 microliters of thesuspension was pipetted into 2960 microliters of 0.1% benzalkoniumchloride (BKC). An intensity of 5×10⁴-1×10⁶ counts/s was achieved. Theparticle size distribution of formulation was measured in triplicate.The average size of the TA particles from Example 9 was in the 300-400nm size range (n=3). See FIG. 32 .

Thermal Characteristics of TA Nanocrystals Vs. TA Stock Material

Thermal properties of the TA particles from Example 9 were investigatedusing a SHIMADZU® DSC-60 and TGA-50.

Approximately 10 mg of sample was analyzed in an open aluminum pan, andheated at scanning rate of 10° C. min⁻¹ from room temperature to 320° C.FIG. 33 shows the differential calorimetry scan of TA API. Peak of theheat of melting is at 289.42° C., with ΔH_(m)83.50 J/g. In comparison,peak of the heat of melting for the nanocrystals produced by the processdescribed in Example 9 is at 275.78° C., with ΔH_(m)=108.45 J/g (FIG. 34). The data suggest that TA nanocrystals are markedly more crystalline,evidenced by a higher heat of melting. Further, the large shift inmelting point for the nanocrystals (compared to the API) suggestsdifferences in the internal crystal structures.

FIGS. 35 and 36 are TGA scans of the TA stock material and the TAnanocrystals respectively. Comparatively, it is clear that the boththese materials have very similar weight loss profiles when heated,indicating that the same molecular bonds are breaking as the substancesare heated. However, as in the DSC profiles there are marked differencesin the onset of each phase of weight loss between the materials,suggesting differences in crystal structure and morphology.

Morphology of TA Nanocrystals Vs. TA Stock Material

Morphology of the TA nanocrystals made in Example 9 was investigatedwith Scanning Electron Microscopy (SEM) (Amray 1000A upgraded with a PGT(PRINCETON GAMMA TECH®) Spirit EDS/Imaging system. Sample was argonsputter coated (HUMMER V® from ANATECH®) with gold (˜200 Å). Sample wasmounted on double side tape. FIGS. 37A and 37B are SEM images of the TAstock material, at two different magnifications. FIGS. 37C-E are SEMimages of TA nanocrystals. As seen in the SEM images, the morphology ofthe nanocrystals prepared by the process of the invention is markedlydifferent than that of the stock material from the manufacturer.

TA Nanocrystals Prepared by the Process of the Invention Maintain theirPurity and Integrity

The measurement of triamcinolone acetonide was adapted from Matysovi etal. (2003), “Determination of methylparaben, propylparaben,triamcinolone”, and the only modification made was the increased runtime to compensate for our longer column used for the assay. Sampleswere run at low concentrations in an effort to amplify any contaminantpeaks vs. TA peaks (an effect seen in fluticasone analysis). Theresulting chromatograms were very clean, with a TA peak elution seen at28.9 minutes. The conditions were:

HPLC System: AGILENT® 1100 with CHEMSTATION® Software

Column: PHENOMENEX LUNA®; C18, 5 μm pore size, 100 Å, Dimensions:250×4.60 mm

Mobile Phase: 40/60 v/v Acetonitrile and HPLC grade water.

Injection Volume: 20 μL

Analysis Time: 30 Minutes

Detection Wavelength: 240 nm

Comparison of the IPLC traces of the TA nanocrystals with those of theTA stock material demonstrated that the nanocrystals produced by theprocess of the invention did not degrade as a result of the process ofthe invention.

Crystal Structure of Triamcinolone Acetonide Produced by the Process ofthe Invention Vs. The Triamcinolone Acetonide Stock Material

The triamcinolone acetonide crystals (i.e., Form B) prepared by themethod of this invention have a different crystalline habit from thestock material, as evidenced by the different XRPD patterns in FIG. 39 .In other words, the triamcinolone molecules within the unit cell arepacked differently from those of the stock material. Similar tofluticasone nanocrystals (Form A), this new morphic form oftriamcinolone can have different physiological properties as compared tothe triamcinolone stock material.

Example 11: Nanocrystal Manufacturing Process-Modified Flow andPurification Process

Experiments were designed to generate process conditions that would: (a)reproducibly generate nanocrystals of cumulants mean size asapproximately 500 nm (±200 nm), (b) reproducibly generate stablecrystals, with stability defined by chemical and physical stability and(c) reproducibly maintain crystal size after purification at highcentrifugal forces.

Several modifications to the flow process described in Example 7 weremade. In particular, a mixing step between crystal formation andannealing was added. Other steps that were added include: (a) dilutionwith a “washing solution” between the annealing and the centrifugationsteps, (b) re-dispersion of the pellet in the washing solution forfurther purification, (c) collection of a pellet and its re-dispersioninto the final formulation composition. Using this modified flowprocess, producing nanosuspension at 0.09% drug at 3500 g/min,commercially relevant volumes of nanosuspension can be manufactured. Theflow reactor was equipped with sanitary fittings, designed to beautoclaved. The steps defined in FIG. 38 led to final production ofhighly pure drug crystals of cumulants mean size of 500 nm (±200 nm).

Role of the Probe Design

Scale-up experiments with the purpose of enhancing efficiency wereperformed with both a standard 1″ sonicating probe with a single activetip at the bottom of the probe and a “bump-stick” probe with multiplesonicating tips on the wand.

Standard Probe Experiments:

Various combinations of fluticasone propionate percentage, flow rates,temperatures, and amplitude of sonication were tested to determine theireffects on mean size of the crystals. Fluticasone propionate percentageranged from 0.224% to 0.229%. The flow rate of phase I ranged from 0 to825 mL/min. The flow rate of phase II ranged from 10 to 900 mL/min. Theflow rate of phase III ranged from 25 to 1400 mL/min. The phase II/phaseI flow rate ratio ranged was 1. The temperatures were 0-22° C. for phaseI, 0-22° C. for phase II, 10-40° C. for phase III. The average phase IIItemperature ranged from 12.5 to 40° C. The amplitude of sonicationranged from 25% to 75% output. The resulting mean size (e.g., d50, ormass median diameter) of the crystals ranged from 0.413 μm to 7 μm.

The highest flow rate of phase I and phase II that yielded particles ofsize d50˜500 nm, was 250 ml/min at all output energies (25% output, 75%output). Higher flow rates (at Phase II/Phase I ratio=1) at 700 ml/minfor Phase I and Phase II led to large particle sizes >7 μm.

Experiments with the bump stick probe demonstrated that higher flowrates of Phase I and Phase II could be achieved, thus enhancing theefficiency of the flow process many-fold. Particle sizes of d50≤500 nmcould be achieved when used in synergy with other parametric variablessuch as choice of buffer, pH of phase II, or sonication output energy.All other experiments described in this Example were performed with thebump stick probe.

Role of Buffer and pH in Phase II

The pH of the phase II affected the particle size. The pH of phase IIwas ˜8, resulting in a pH of ˜7 post-mixing of phase I and phase II.Ascorbic and Citrate buffers at pH 4 and pH 5 were investigated asbuffers for phase II. Particle size was measured using a MALVERN® S90®.The MALVERN® S90® measures particle size by dynamic light scattering(DLS). For needle-shaped crystals as the proprietary fluticasonepropionate crystals produced by this process, the most relevant valuefor particle size measured by DLS is the peak mean, or the cumulantsmean. Thus, all particle size values are reported as the cumulants mean.An example is shown in Table 17.

TABLE 17 Particle Size (cumulants peak mean) as a Function of AscorbicAcid Buffer, pH 5 Particle Particle Particle Time Size (nm)₁ Size (nm)₂Size (nm)₃ 25° C. Annealing t0 (post titration) 748.90 768.10 678.60 t1(+98 hours) 596 510.8 509.2 40° C. Annealing t0 (post titration) 748.90768.10 678.60 t1 (+98 hours) 596.9 441.8 766.3

Both 25° C. and 40° C. are suitable as annealing temperatures.Additional temperatures may also be suitable for annealing. Ascorbatebuffer, pH 5 used in phase II generated particles between 500-800 nm(d50). Citrate buffer at pH 4 and pH 5 were investigated as thebuffering agent in phase II in multiple flow reactor batches.Representative examples are shown in Tables 18-19.

TABLE 18 Particle Size (cumulants peak mean) as a Function of CitrateBuffer, pH 4 Particle Particle Particle Time Size (nm)₁ Size (nm)₂ Size(nm)₃ t1 pre-mixing 476.1 510.2 610.6 t2 after 30 m mix 588.5 617.1465.7

TABLE 19 Particle Size (d₅₀) as a Function of Citrate Buffer, pH 5Particle Particle Particle Time Size (nm)₁ Size (nm)₂ Size (nm)₃ t0pre-mixing 630.4 625.6 654.5 t1 after 30 m mix 624.7 516.4 645.5

In general, both citrate and ascorbate buffers were suitable, andstatistically, no differences were noted. Citrate buffer was selected asthe buffer of choice due to its presence in multiple pharmaceuticalformulations. pH 5 was selected as the pH of choice of phase II, due toslight increases in impurities shown in nanosuspensions prepared at pH 4and annealed at 25° C. Nanosuspensions prepared in phase II at pH 5,citrate buffer showed no increase in impurities during annealing.

Role of Sonication Output Energy

The sonication output energy was investigated as a variable in thegeneration of nanocrystals of particle size with a cumulants mean valueat 500 nm (±200 nm). To obtain detailed statistically meaningful data onparticle size, a HORIBA® LA-950 Laser Diffraction Particle Sizer wasutilized, which provides the statistical mean, median and mode of eachbatch analyzed.

Table 21 is an example of a batch prepared at 40% output energy, 1:4Phase I: Phase II ratio. The composition of phase II was 0.4% 15centipoise methyl cellulose (MC), 0.005% benzalkonium chloride, 0.1%PEG40 Stearate in citrate buffer, pH 5 and distilled water. The datashown in Tables 22-24 are representative of batches produced at 50%, 60%and 70% output energies, with all other parameters identical, or assimilar as possible. Thus, the phase I, phase II and phase IIIcompositions were the same, temperatures of each of the phases in eachbatch were similar, as well as the temperatures of annealing. Theannealing temperature of the incubator ranged from 25-28° C., with a65%-75% relative humidity. The flow rate of Phase III for each of thebatches was 3250 g/min (±200 g/min). After production of thenanocrystals, each batch was mixed with a SCILOGIX® mixer at 250 RPM atroom temperature. Batch sizes were approximately 3500 grams. Phase IIIcomposition of each batch is tabulated in Table 20.

TABLE 20 Phase III Composition with a 1:4 Phase I:Phase II RatioComponent grams % Fluticasone Propionate 3.15 0.09 TWEEN 80 ®(polysorbate 80) 53.13 1.52 Polypropylene Glycol 400 481.67 13.762Polyethylene Glycol 400 162.05 4.63 Methyl Cellulose 15 cP 11.2 0.32PEG40 Stearate 2.8 0.08 Benzalkonium Chloride 0.14 0.004 Citrate Buffer(0.1M), pH 5 44.8 1.28 Water 2714.06 78.32

Particle size data was provided in terms of mean, median and mode. Bydefinition, the mode particle size signifies the highest number ofparticles with that size, the median particle size signifies the numberof particles that are in the “middle” of the distribution, and the meanparticle size is the average of all sizes for the entire distribution.For a perfect monomodal Gaussian distribution, the mean, median and modeare all similar. In distributions that are skewed, these values differwidely. The mean, median and mode values are all within a 250 nm range,after at least 24 hours of annealing.

TABLE 21 Representative batch prepared with 40% output energy 1:4 Batch,pH 5, 40% amplitude Flow rate: Amp: 40% Mean Median Mode d90 d50 Time(um) (um) (um) (um) (um) t0 0.67215 0.44926 0.3638 1.4063 0.4493 PEG-(+)30 0.6827 0.44473 0.3632 1.4527 0.4447 Stearate min mix 0.1% (+)240.7038 0.44397 0.3629 1.5136 0.444

TABLE 22 Representative batch prepared with 50% output energy 1:4 batch0.005% BKC, 0.1% PEG-Stearate, pH 5 phase II. Phase III temp = 11.5;rate = 3495 g batch size; 50% AMP Flow rate: 3608 g/min Amp: 50% TimeMean Median Mode d90 d50 (hrs) (um) (um) (um) (um) (um)  0 0.599840.43397 0.3647 1.179 0.434 PEG- (+)30 0.56879 0.40337 0.3619 1.16720.4034 Stearate min mix 0.1%  96 0.61444 0.41099 0.3618 1.2931 0.411 1120.64135 0.4125 0.3616 1.3758 0.4125

TABLE 23 Representative batch prepared with 60% output energy 1:4 batch0.005% BKC, 0.1% PEG-Stearate, pH 5.04 phase II. 13° C. Phase III-3498 gbatch. 60% Amp. Flow rate: Amp: 60% Mean Median Mode d90 d50 Time (um)(um) (um) (um) (um) t0 0.72887 0.54961 0.4781 1.3807 0.5496 PEG- (+)300.71239 0.51732 0.4172 1.429 0.5173 Stearate min mix 0.1% (+)24 0.694010.52177 0.418 1.3659 0.5218 (+)48 0.76579 0.52094 0.4173 1.5413 0.5209(+)144  0.6936 0.51772 0.4181 1.348 0.5177 (+)144  0.75277 0.522470.4176 1.5225 0.5225

TABLE 24 Representative batch prepared with 70% output energy 1:4 batch0.005% BKC, 0.1% PEG-Stearate, pH 5 phase II. Phase III temp = 13; rate= 3470; g batch size; 70% Amp Flow rate: 3495 g/min Amp: 70% Mean MedianMode d90 d50 Time (um) (um) (um) (um) (um) t0 2.93615 0.43001 0.36312.9617 0.43 PEG- (+)30 0.65677 0.4636 0.38867 1.5569 0.3887 Stearate minmix 0.1% (+)96 0.52345 0.40234 0.363 1.0063 0.4023 (+)112  0.5985 0.39350.3611 1.2603 0.3936

TABLE 25 Representative batch prepared with 80% output energy 1:4 batch,pH 5, 80% amplitude Flow rate: Amp: 80% Mean Median Mode d90 d50 Time(um) (um) (um) (um) (um) t0 0.88407 0.34681 0.1836 2.2933 0.3468 PEG-(+)30 1.19832 0.56541 0.3645 2.8992 0.5654 Stearate min mix 0.1% (+)241.61358 0.57793 0.365 3.4731 0.5779

Thus, initial particle size (T=0 values) generated by crystallization inthe presence of sonication is almost directly correlated to outputenergy, i.e. the higher the output energy, the smaller the statisticalmode (the most frequently occurring size).

By annealing, the particles can settle into a lower energy state. Theparticles have high surface energy with increase in output energy,causing the particles to agglomerate. This is evidenced in Table 24,which describes particle size dynamics of a batch generated with 70%output energy. At T=0, the batch had a mean particle size of 2.93microns and a mode value (“most frequent” value) of 0.3631 microns,indicating that even if most of the particles were <500 nm, there weresome large particles in the distribution that skewed the mean. At T=96hours of annealing at 25° C., the mean, median and mode were within 250nm of each other, proving that the larger particles skewing the meanwere agglomerates. Annealing the batch lowered the surface energy intoan equilibrated ground state, thus de-aggregating the particles.

Particle Size Decreases with Annealing

Annealing has been shown to be a critical part of the batch process, asshown in previous data. Annealing of crystals generated by thecontinuous flow process has also proved to be a significant part of theprocess, as discussed in the previous section.

It was also demonstrated above that the kinetics of annealing isimportant. In various experiments, it did seem that particle sizes ofbatches annealed at 25° C., 40° C. and 60° C. did not significantlydiffer from each other in terms of particle sizes. However, annealinghas another purpose. The crystallization can be “completed” byannealing, thus “hardening” the crystals. From this perspective, thehigher the temperature of annealing without degradation, the morecrystalline the particles will be.

Table 26 shows a batch prepared with the ascorbate-buffered phase II, pH5, annealed at two different temperatures. These batches were preparedwith ascorbic acid buffer, pH 5, phase I: phase II: 1:3, 60% outputenergy. The particle size was measured by the MALVERN® S90®. The samebatch of particles annealed at two different temperatures show adifferent mean peak size, as measured by the instrument. However, bothsets show decrease in particle size with annealing.

TABLE 26 Representative Batch Annealed at 25° C. 25° C. AnnealingParticle Size Particle Size Particle Size Time d_(50,) (nm)₁ d_(50,)(nm)₂ d_(50,) (nm)₃ t0 (post titration) 748.90 768.10 678.60 t1 (+98hours) 596 510.8 509.2

TABLE 27 Representative Batch Annealed at 40° C. 40° C. AnnealingParticle Particle Particle Time Size (nm)₁ Size (nm)₂ Size (nm)₃ t0(post titration) 748.90 768.10 678.60 t1 (+98 hours) 296.9 441.8 766.3

Role of Mixing Head Design

The design of the mixing head is important for mixing the nanosuspensionright after crystallization in the flow reactor. The mixing heads weretested in multiple experiments. SILVERSON® mixing heads were evaluated.The medium and the low shear mixing heads (co-axial and paddle) providedthe best particle sizes. The paddle mixer was selected as the mixinghead of choice for all batches.

Role of Benzalkonium Chloride

Benzalkonium chloride is needed to generate particles with a statisticalmode value of ˜500 nm.

Table 28 is a representative batch that was prepared with nobenzalkonium chloride in phase II. The mean, median and mode valuevariance was within 250 nm. The mode value was 1.07 microns. Sinceparticle sizes of ˜1 micron and greater were obtained for all batchesproduced with no benzalkonium chloride, it is deemed necessary forbenzalkonium chloride to be present in phase II, in order to generateparticles of sizes with a statistical mode of ˜500 nm. Batches describedin Tables 28, 29, 30A, and 30B were analyzed with the HORIBA® LA-950®Laser Diffraction Particle Size Analyzer.

TABLE 28 1:4 batch w/ no BAK; pH 5.21 phase II-14° C. phase III-4346.87g/min, 3332.6 g batch. 60% Amp. Phase III Flow rate: 4346.87 g/min MeanMedian Mode d90 d50 Time (um) (um) (um) (um) (um) 1.16041 0.85161 1.07822.3984 0.8516 (+) 30 min mix 1.22985 0.9466 1.2295 2.4875 0.9466 (+) 60hrs 1.24168 0.93764 1.2274 2.5123 0.9376

Table 29 is a representative batch prepared with 20 ppm (0.002%)benzalkonium chloride in phase II. Phase II was also buffered withcitrate, pH 5. The flow rate of phase III was 3250±200 nm. The batch wasa 1:4 ratio batch. Thus, the BAK concentration in phase III was 16 ppm.The batch meets the T=0 particle size specification of statistical mode<500 nm (±200 nm).

TABLE 29 1:5 batch with 0.002% BKC, pH 5.0 Phase II; 11° C. Phase III;3369.2 g batch; 60% Amp Flow rate: 3369.5 g/min Amp: 60% Mean MedianMode d90 d50 Time (um) (um) (um) (um) (um) 0 hrs 0.54942 0.45566 0.4160.9908 0.4557 0 hrs (+) 30 0.41045 0.26352 0.2104 0.9282 0.2635 min mix(+) 15 hrs 0.58982 0.46256 0.3658 1.1239 0.4626 (+) 48 hrs 0.72260.45139 0.3636 1.5348 0.4514 (+) 72 hrs 0.63121 0.43998 0.3628 1.29780.44

Table 30A and 30B are representative batches prepared with 50 ppm(0.005%) benzalkonium chloride in phase II. Phase II was also bufferedwith citrate, pH 5. The flow rate of phase III was 3250±200 nm. Thebatch was a 1:4 ratio batch. Thus, the BAK concentration in phase IIIwas 40 ppm. The batches meet the T=0 particle size specification ofstatistical mode <500 nm (±200 nm). These batches also containedPEG40-stearate as a stabilizing molecule.

TABLE 30A 1:4 batch 0.005% BKC, 0.1% PEG40-Stearate, pH 5.04 phase II.13° C. Phase III-3498 g/min, 60% Amp. Flow rate: 3250 Amp: 60% MeanMedian Mode d90 d50 Time (um) (um) (um) (um) (um) t0 0.72887 0.549610.4781 1.3807 0.5496 PEG- (+)30 0.71239 0.51732 0.4172 1.429 0.5173Stearate min mix 0.1% (+)24 0.69401 0.52177 0.418 1.3659 0.5218 (+)480.76579 0.52094 0.4173 1.5413 0.5209 (+)144  0.6936 0.51772 0.4181 1.3480.5177 (+)144  0.65277 0.52247 0.4176 1.5225 0.5225

TABLE 30B 1:4 batch 0.005% BKC, 0.1% PEG-Stearate, pH 5 phase II. PhaseIII temp = 11.5; rate = 3495 g/min; 50% AMP Flow rate: 3608 g/min Amp:50% Time Mean Median Mode d90 d50 (hrs) (um) (um) (um) (um) (um)  00.59984 0.43397 0.3647 1.179 0.434 PEG- (+)30 0.56879 0.40337 0.36191.1672 0.4034 Stearate min mix 0.1%  96 0.61444 0.41099 0.3618 1.29310.411 112 0.64135 0.4125 0.3616 1.3758 0.4125

Role of PEG 40-Stearate

0.0100 PEG40-stearate was used as the sole stabilizer in acitrate-buffered phase II, 1:3 Phase I/phase II ratio, 60% AMP. Thisdata was analyzed by the MALVERN® S90®. The particle size shown is thecumulants mean. As shown in Table 31, the particle size specification ofmeeting the cumulants mean of 500 nm was met. The level ofPEG40-stearate will vary depending on if a benzalkonium chloride-freebatch is prepared.

TABLE 31 0.01% PEG-Stearate phase II, Mixed w/ paddle mixer @ 250 rpmfor 30 m. Final pH = 5.47 25° C. Annealing Particle Particle ParticleTime Size (nm)₁ Size (nm)₂ Size (nm)₃ t0 556.1 665.1 582.2 t1 + 68 hours554.7 863.7 426.6

Fluticasone Propionate Nanocrystals Purified by Continuous FlowCentrifugation

Continuous Flow Centrifugation was demonstrated as the preferred meansof purifying the crystals. Through purification, the continuous phase ofphase III is centrifuged out. The pellet is re-dispersed as aconcentrate in the washing solution and the dispersion re-centrifuged.Continuous centrifugation was performed by a SORVALL® Contifuge or aBECKMAN COULTER® JI-30 with a JCF-Z® Rotor can be used.

In general, after the nanosuspension has been annealed overnight, thebatch is then diluted 1:1 with 0.1% PEG40-Stearate, 0.1% TWEEN 80®(polysorbate 80) and 50 ppm Benzalkonium Chloride. Dilution of thenanosuspension lowers the viscosity of phase III to enable ease ofcentrifugation.

The BECKMAN® centrifuge is cooled to 4° C., and the suspensioncentrifuged at 1.6 L/min at 39,000 G. The supernatant appeared clear anddevoid of particles. The particle size distributions are shown in Table32. This batch had been prepared with no benzalkonium chloride. Thus,the particle size is larger than the usual 500 nm statistical mode.Surprisingly, after purification, the mode shifts to <500 nm. This showsthat the centrifugation breaks down agglomerated particles. This is away to eliminate large particles.

TABLE 32 1:4 batch w/ no BKC; pH 5.21 phase II-14° C. phase III-4346.87g/min, 3332.6 g batch. 60% Amp. Flow rate: 4346.87 g/min Mean MedianMode d90 d50 Time (um) (um) (um) (um) (um) 1.16041 0.85161 1.0782 2.39840.8516 (+) 30 min 1.22985 0.9466 1.2295 2.4875 0.9466 mix (+) 60 hrs1.24168 0.93764 1.2274 2.5123 0.9376 purified after 1.1979 0.739980.4747 2.6483 0.74 60 hrs

Flow process variables that play a role in particle size aretemperatures of phase I and phase II, pH of phase II, composition ofphase II, output energy, probe design, flow rate, ratio of phase II tophase I, annealing temperature, mixing conditions after particleproduction and composition of washing solution prior to purification.These results demonstrate for the first time that the manufacturing flowprocess produces commercial volumes of fluticasone propionatenanosuspension crystals and that the crystals can be purified using highflow continuous centrifugation.

Example 12: Formulations of FP Nanocrystals and Evaluation

Formulations containing fluticasone propionate nanocrystals withdifferent FP contents (e.g., 0.25%±0.0375% (0.21-0.29%), 0.1%±0.015%(0.085-0.115%), and 0.05%±0.0075% (0.043-0.058%)) were prepared andevaluated. The following parameters of each formulation were evaluated:spreading of formulation on the skin (minimum contact angle preferred),chemical compatibility (of other ingredient) with FP, dose uniformityand redispersibility, stability of particle (e.g., unchanged size ofparticles preferred), and droplet size (function of viscosity andintermolecular surface tension, maximizing droplet size preferred).

Tables 33 and 34 below list the components of two differentpharmaceutical formulations (each containing 0.25% FP) that wereprepared for use in treating, e.g., blepharitis.

TABLE 33 Formulation I Composition Ingredients (%) Intended FunctionFluticasone Propionate 0.250 Active Benzalkonium chloride 0.005Preservative Polysorbate 80 0.200 Coating Dispersant Glycerin 1.000Tissue Wetting Agent PEG stearate 0.200 Coating Dispersant Methylcellulose 4000 cP 0.500 Polymeric stabilizer Sodium Chloride 0.500Tonicity Adjustment Dibasic sodium phosphate 0.022 Buffering AgentMonobasic sodium phosphate 0.040 Buffering Agent Water 97.340

TABLE 34 Formulation II Composition Ingredients (%) Intended FunctionFluticasone Propionate 0.250 Active Benzalkonium chloride 0.005Preservative Glycerin 1.000 Tissue Wetting Agent Tyloxapol 0.200 CoatingDispersant Methyl cellulose 4000 cP 0.500 Polymeric stabilizer SodiumChloride 0.500 Tonicity Adjustment Dibasic sodium phosphate 0.022Buffering Agent Monobasic sodium phosphate 0.040 Buffering Agent Water97.483

Formulation I, ingredients of which are listed in Table 33 above, wasevaluated and had the following properties: Viscosity=45±4.1 cP;pH=6.8-7.2; osmolality=290-305 mOsm/kg; particle size: statistical mode:400 nm, median: 514 nm, mean: 700 nm, d50: 400 nm, d90: 1.4 μm; anddroplet size=40±2 μL. Further, Formulation I was redispersible uponshaking, exhibited uniform dose for at least one hour after shaking; andthe particle size was stable for at least 21 days at a temperaturebetween 25° C. and 40° C.

Formulation II, ingredients of which are listed in Table 34 above, wasevaluated and had the following properties: Viscosity=46±3.2 cP;pH=6.8-7.2; osmolality=290-305 mOsm/kg; particle size: statistical mode:410 nm, median: 520 nm, mean: 700 nm, d50: 520 nm, d90: 1.4 μm; anddroplet size=40±2.3 μL. Further, Formulation II was redispersible uponshaking, exhibited uniform dose for at least one hour after shaking; andthe particle size was stable for at least 18 days at a temperaturebetween 25° C. and 40° C.

Average droplet sizes of other formulations having different FP contents(i.e., about 0.25%, 0.1%, 0.05%, and 0%) were tested are summarized inTable 35 below. The test was conducted using a 7 mL drop-tip eye-dropperbottle with a 5 mL fill and with drop-tip pointed vertically down. Theamount of FP per droplet was determined by HPLC.

TABLE 35 0.25% FP 0.1% FP 0.05% FP 0% FP ave. droplet FP per ave.droplet FP per ave. droplet FP per ave. droplet FP per size (μL) drop(μg) size (μL) drop (μg) size (μL) drop (μg) size (μL) drop (μg) 41.17102.925 ± 39.54 39.54 ± 40.65 20.325 ± 40.27 0 3.5766 3.1263 1.950

As shown in Table 35 above, the droplet size was consistent across allof the formulations tested.

To test drug delivery efficiency of different applicators, the 0.25 FP %Formulation I mentioned above was loaded to various applicators such asswabs and brushes (e.g. FOAMEC-1® swab, polyurethane swab, polyesterswab, 25-3318-U swab, 25-3318-H swab, 25-3317-U swab, 25-803 2PD swab,25-806 1-PAR swab, cotton swab, and LATISSE® (bimatoprost opthalmicsolution) brush), and then each FP-loaded applicator was swiped againsta polypropylene membrane to determine how much FP was transferred ontothe membrane.

More specifically, for each applicator, two drops of Formulation I wereloaded on the applicator before swiping the applicator on apolypropylene membrane twice. The FP transferred onto membrane was thenextracted with the mobile phase used for HPLC analysis to determine theamount of FP transferred onto the membrane. For each kind of applicator,the same measurement was repeated 3-8 times. It was observed thatLATISSE® (bimatoprost ophthalmic solution) brushes demonstrated betterdrug delivery (i.e., about 56% FP transferred on average) topolypropylene membrane than the other applicators. Ranked the second was25-3317-U swab (i.e., about 34% FP transferred on average). The averagepercentage of FP delivered to the polypropylene membranes by each of theother applicators tested is listed in Table 36 below.

TABLE 36 25- 25- 25- Poly- 3318- 3318- 25- 8061- Cotton Foamec-1urethane Polyester U H 8032PD PAR swab 6.9-22.17 1.06 0.41 13.92 18.7114.39 1.03 0.94

It was also observed that polyester swabs and cotton swabs absorbed theformulation drops quickly; and when swiped on membrane, the FP wasbarely transferred. On the other hand, polyurethane swabs “beaded” thedrops-drops fell off. It took two seconds for LATISSE® (bimatoprostophthalmic solution) brush to absorb 1^(st) drop and 1.3 seconds for25-3317-U swab to absorb 1^(st) drop. In terms of ease of use, LATISSE®(bimatoprost ophthalmic solution) brushes are easier to use compared tothe other applicators tested.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. While specificembodiments of the subject invention have been discussed, the abovespecification is illustrative and not restrictive. Many variations ofthe invention will become apparent to those skilled in the art uponreview of this specification. The full scope of the invention should bedetermined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations. Suchequivalents are intended to be encompassed by the following claims.

What is claimed:
 1. A method of alleviating at least one symptomassociated with blepharitis comprising administering to a subject inneed a therapeutically effective amount of a topical formulationcomprising a suspension of 0.001% to 5% w/v nanocrystals of fluticasonepropionate polymorph 1 having a crystalline habit (Form A) characterizedin that a [001] crystallographic axis is substantially normal tosurfaces that define a thickness of nanoplates, wherein the nanocrystalshave an average size of 100 nm to 1000 nm and a X-ray powder diffractionpattern with characteristic peaks at about 7.8, 15.7, 20.8, 23.7, 24.5,32.5 degrees 2θ and additional peaks at about 9.9, 13.0, 14.6, 16.0,16.9, 18.1, and 34.3 degrees 2θ and a pharmaceutical acceptable aqueousvehicle, wherein the method comprises administering to eyelid, eyelashes, or eyelid margin of subject in need a therapeutically effectiveamount of said topical formulation, wherein the at least one symptom isselected from inflammation of eyelid margin, eyelid redness, eyelidswelling, eyelid discomfort, eyelid itching, flaking of eyelid skin,ocular redness, and a combination thereof.
 2. The method according toclaim 1 wherein the topical formulation further contains one or morecoating dispersants, one or more tissue wetting agents, one or morepolymeric stabilizers, one or more buffering agents, and/or one or moretonicity adjusting agents.
 3. The method according to claim 2 whereinthe topical formulation further containing about 0.002% to 0.01% w/v ofbenzalkonium chloride.
 4. The method according to claim 1 wherein aconcentration of nanocrystals of fluticasone propionate is 0.01% to 1%w/v.
 5. The method according to claim 1 wherein a concentration ofnanocrystals of fluticasone propionate is 0.25% w/v.
 6. The methodaccording to claim 1 wherein a concentration of nanocrystals offluticasone propionate is 0.1% w/v.
 7. The method according to claim 1wherein a concentration of nanocrystals of fluticasone propionate is0.05% w/v.
 8. A method of alleviating at least one symptom of diseasesassociated with meibomian gland dysfunction (MGD) comprisingadministering to a subject in need a therapeutically effective amount ofa topical formulation comprising a suspension of 0.001% to 5% w/vnanocrystals of fluticasone propionate polymorph 1 having a crystallinehabit (Form A) characterized in that a [001] crystallographic axis issubstantially normal to surfaces that define a thickness of nanoplates,wherein the nanocrystals have an average size of 100 nm to 1000 nm and aX-ray powder diffraction pattern with characteristic peaks at about 7.8,15.7, 20.8, 23.7, 24.5, 32.5 degrees 2θ and additional peaks at about9.9, 13.0, 14.6, 16.0, 16.9, 18.1, and 34.3 degrees 2θ and apharmaceutical acceptable aqueous vehicle, wherein the method comprisesadministering to eyelid, eye lashes, or eyelid margin of subject in needa therapeutically effective amount of said topical formulation, whereinthe at least one symptom is selected from inflammation of eyelid margin,dry eye, eye redness, itching and/or irritation of eyelid margins,edema, foreign body sensation, matting of lashes, and a combinationthereof.
 9. The method according to claim 8 wherein the topicalformulation further contains one or more coating dispersants, one ormore tissue wetting agents, one or more polymeric stabilizers, one ormore buffering agents, and/or one or more tonicity adjusting agents. 10.The method according to claim 9 wherein the topical formulation furthercontaining about 0.002% to 0.01% w/v of benzalkonium chloride.
 11. Themethod according to claim 8 wherein a concentration of nanocrystals offluticasone propionate is 0.01% to 1% w/v.
 12. The method according toclaim 8 wherein a concentration of nanocrystals of fluticasonepropionate is 0.25% w/v.
 13. The method according to claim 8 wherein aconcentration of nanocrystals of fluticasone propionate is 0.1% w/v. 14.The method according to claim 8 wherein a concentration of nanocrystalsof fluticasone propionate is 0.05% w/v.